Chemistry 1 - Lab Practical 1 Flashcards
Identify the four basic measurement techniques seen in the modern medical laboratory.
Spectrometry
- Spectrophotometry
- Atomic Absorption
- Mass Spectrometry
Luminescence
- Florescence
- Chemiluminescense
- Nephelometry
Electroanalytical methods
- Electrophoresis
- Potentiometry
- Amperometry
Chromatography
- Gas
- Liquid
- Thin-layer
Differentiate photometry and spectrophotometry.
Photometry - Based on the principle that radiant energy that passes through an object will be partially reflected, absorbed and transmitted.
Spectrophotometry – utilizes filters or prisms to isolate a narrow wavelength of radiant energy and a detector to convert transmitted light into an electrical signal.
Diagram the basic components of a spectrophotometer.
Light source -
> Tungsten or tungsten-iodine lamp (visible and near IR region, wide spectrum ~320 to >1000 nm).
> Mercury-arc or deuterium-discharge (UV light, narrower spectrum).
> Lasers (very narrow bandwidths, high intensity).
Monochromator -
> Filter = Colored glass, one or many layers, Transmits only a small range of wavelengths, Number on filter indicates peak transmittance.
> Prism = Separates the wavelengths, Rotate the prism to select the desired, wavelength
> Grating = Uses diffraction to separate wavelengths, Slit selects the wavelength
Sample (Cuvette)
Light Detector
Readout Device
Assess the function of the basic components of a spectrophotometer.
Light source - Light is a type of radiant energy that is visible to the human eye (travels in waves, visible to human eye ~400 – 700 nm).
Wavelength (λ) (distance between points on the wave, inversely proportional to energy, measured in nanometers (nm = 1x10-9m).
Monochromator - A system that isolates radiant energy of desired wavelength and excludes other wavelengths.
> Spectral bandpass = the range of wavelengths that will be transmitted.
> Calculated as width (nm) of the spectral transmittance curve at a point equal to one-half (50%) the peak transmittance.
> Usually composed of two parts: Wavelength splitter to split the spectrum & Slit to select portion of spectrum.
Sample (Cuvette) - Holds sample in the light path.
> Must be constant light path (flow through cells eliminate cuvette variability).
> Should be as transparent as possible to the light being used (e.g. visible, uv). Cuvette material depends on wavelength being measured.
> Surfaces should be clean. Particles and fingerprints absorb or scatter the light.
> Do not use scratched cuvettes. Scatter light.
Light Detector -
> Light energy is converted to electrical signal.
> Photoelectric cell (photomultiplier tube) – a sensitive instrument that produces electrons in proportion to the amount of light hitting it.
Readout Device -
> Electrical signal is passed to a readout device called a galvanometer.
> Galvanometer records current (electrons) received on a viewable scale. (May be digital or analog (Pointe 180–digital, Spectronic 20–analog).
Examine the nature of light including how it is measured and its relationship to energy.
- Light is a type of radiant energy that is visible to the human eye
> Travels in waves
> Visible to human eye ~400 – 700 nm - Wavelength (λ)
> Distance between points on the wave
> Inversely proportional to energy
> Measured in nanometers (nm = 1x10-9m)
Given a wavelength of light, predict the color seen and absorbed.
< 380 = UV, Not visible
380-440 = Violet
440-500 = Blue
500-580 = Green
580-600 = Yellow
600-620 = Orange
620-750 = Red
750-2000 = IR, Not visible
Define spectral bandpass.
The range of wavelengths that will be transmitted.
Calculated as the width (nm) of the spectral transmittance curve at a point equal to 1/2 (50%) the peak transmittance.
Compare types of monochromators.
Monochromator -
> Filter = Colored glass, one or many layers, Transmits only a small range of wavelengths, Number on filter indicates peak transmittance.
> Prism = Separates the wavelengths, Rotate the prism to select the desired, wavelength
> Grating = Uses diffraction to separate wavelengths, Slit selects the wavelength
Evaluate quality control testing on spectrophotometers to determine acceptability.
- Wavelength accuracy
> Read standard materials
> Results must be within the specified tolerances - Stray light
> Any light outside the band sent by the monochromator
> Sources of stray light (Scratches on cuvette or lenses, Higher-order spectra from gratings). - Linearity
> A change in concentration results in a proportional change in the measurement (Must result in a straight line plot).
> Standard filters for linearity are available
> Standard readings must meet specs
Contrast absorbance and percent transmittance.
Absorbance =
> An expression of the amount of light absorbed by a solution.
> How do you measure absorbance directly? You must measure the TRANSMITTED light.
Percent Transmittance =
> The amount of light that passes through a colored solution compared with the amount of light that passes through a blank or standard solution.
Convert between absorbance and percent transmittance.
A logarithmic relationship exists between %T and concentration.
Absorbance is equal to 2 minus the log of percent transmittance.
A = 2-log %T
Example:
What is the absorbance for a %T of 80?
A = 2 – log 80
A = 2 – 1.9
A = 0.1
State Beer-Lambert’s Law.
The concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light.
A = ebc
ε = Molar absorptivity
> Often expressed as ‘a’
> The absorbance per cm of a 1M solution
> Constant for each specific compound
b = path length (must be constant)
c = concentration
Perform calculations using Beer-Lambert’s Law.
Assumptions:
> Light being measured comes only from the analytical beam of light
> Light is monochromatic
(only one wavelength)—no stray light
> Substance being analyzed is the only colored solute (chromophore) present in the solution
Standard As = (aS)b(cs) Unknown Au = (au)b(cu)
aS(b) = au(b)
As/cs = Au/cu
cs/As = cu/Au
(cs x Au)/As = cu
Predict concentration of unknown from Standard Calibration Curve.
- Absorbance vs. concentration
> Results in a straight line on linear graph paper - %Transmittance vs. concentration
> Makes straight line on semi-log paper
Resolve case studies involving spectrophotometry.
Do practice questions slide 42-47
Describe relationships in absorbance spectrophotometry.
Intensity of color is directly proportional to the concentration of the solution
Concentration of an unknown
> Determined by measuring its absorbance of light at a particular wavelength
> Compare it’s absorbance to that of the same light by a known standard.
Several standards of varying concentrations can be determined and then plotted on graph paper.
The resulting graph is called a standard calibration curve.
For electrolyte methodology, what is the most popular specimen used for Na+ and K+ detection?
- Serum (most popular)
- Plasma (need to know anticoagulant (no EDTA))
- Whole Blood (capillary) - must warm up capillary blood to 40*C to stimulate vasodilation (capillaries become more like arterial samples and less like venous samples under certain conditions).
- Urine
- Body Fluid (CSF)
- Feces
- GI fluid
What are sources of error for Na+ and K+ measurement?
- Lipemic blood (fat): drawing blood after eating may lead to blood with fatty components, which could elevate spectrophotometry.
- Hemolysis: K+ (ions in serum of plasma could be artificially elevated, even in slight hemolysis).
- Storage Temperature: 4C vs 37C (Store at LOWER temp!!! Enzymes are more active at higher temp, which increases O2 use, causes hypoxia, and then cell death and hemolysis).
- Timing of Separation of plasma: don’t want cells to become acidic and cause cell death.
What are methods for detecting Na+ and K+?
- Ion Selective Electrodes: you have reference vs. test electrodes that detect the # of ions in a water fraction. Na+ uses a glass membrane electrode. K+ uses a valinomycin coated polyvinyl chloride electrode.
> Direct (undiluted) - not effected by hyperlipidemia. The value is what it is.
> Indirect (diluted) - effected by hyperlipidemia (fat in blood). Can look like pseudo-hyponatremia (fake). (Ex. 145 mM in 1000 mL water is exact is using direct method. In indirect method, same sample could read as 130 mM in 1000 mL of water. A corrective calculation needs to be done depending on water concentration. If 10% of the 1000 mL water in an indirect method are fat, then the correction is — 130:900 : x:1000. x = 144.44 mM in 100 mL solution via correction, to there really is no hyponatremia. - Flame Atomic Emission Spectroscopy: highly sensitive and accurate where the intensity of emitted light is proportional to ion concentration of a sample. Involves a monochromator (selects specific wavelengths of light) and a photodetector (detects specific wavelengths of light).
> Can’t use direct method for this. Must use indirect method (means it requires dilution and you have to worry about interference of increased protein (pseudohyponatremia/pseudo-hypochloremia. K+ is less effected)).
> Not popular for measuring basic ions, but can be used to detect Ca2+ and Lithium.
Wavelengths - Na+: 589 nm / K+: 766nm / Ca2+: 852 nm / Li+: 671 nm
Describe specimen collecting and handling for Na+ and K+.
- Specimens: serum, heparinized plasma + whole blood, urine, and other fluids.
- Plasma used for K+ detection (if you use the serum, K+ can be squeezed out of cell during clotting, which you need to let serum clot for this).
> *Note: Plasma is the liquid you get with added anticoagulants. Serum is the liquid you get when clotting factors and RBCs are clotted out of the liquid. - Don’t use Na+ heparin/NH4- heparin (this causes an increase in H+ concentration > acidic conditions > hemolysis > leakage of ions).
- Stability
- Whole blood should be analyzed within 3 hours (cell death/hemolysis).
- Plasma, serum, urine Na+/K+ (can store for 1 week at room temperature & 1 year at -20*C.
Methods for detecting Cl-
Interference with other halides
- Coulometric Titration - preferred method & very accurate. Uses silver (Ag) electrode, where this electrode releases the silver (Ag) and it reacts with Cl- to make AgCl. When all the Cl- is used up (indicated by free Ag+), timer is stopped. Duration of time can be used to figure out how much Cl- is in the patient sample. The more Cl- present, the longer the process.
> Problem: excess AgCl production. An insoluble precipitation creates problems in the automatic analyzers. - Mercuric Thiocyanate Spectroscopy - good method for Cl- in normal range (80-125 mM). A very temperature sensitive method. Wrong temperature could cause unstable readings. Uses ferric-isothiocyanate.
> Interfering factors: bilirubin, Hgb, lipemia
> Problem: signal noise at lower [Cl-]. It can make a lot of noise. - Ion Selective Electrodes - most common method. Direct and indirect methods (same applications as with Na+/K+. Has silver (AgX) complex sensing capabilities.
- Schales and Schales Titration - chloride ions in serum are complexed with mercury (Hg[NO3]2 (known concentration)) to form soluble HgCl2.
> Serum proteins are precipitated with tungstic acid to remove the proteins from the sample. Then after the proteins are removed, the soluble fraction is titrated with mercuric nitrate in the presence of s-diphenylcarbazone as indicator (turns violet-blue with first excess of mercuric ion).
> Bilirubin or Hgb may obscure the endpoint.
Specimen collection & handling for Cl-:
Serum, heparinized plasma, urine, sweat (CF - newborn baby), & other body fluids.
All fluids should rapidly be separated from cells to avoid pH changes and shift in ionic equilibria due to cellular metabolism. (Cl- & HCO3- are related, HCO3- & pH are related).
Stability:
Chloride in serum, plasma, urine, and other fluids are stable for 1 week at RT and for 2 weeks at 2 & 20*C.
ISE measurements of whole blood should be performed within 2 hours.
Sweat Chloride:
Important test for cystic fibrosis (defect in CFTR (cystic fibrosis transduction regulator) regulator proteins), generally administered to children (newborn baby screening test (common in caucasian populations)). .
Induce sweating with pilocarpine.
Typically, ISE method used
Normal: 0-40 mmol/L
Cystic fibrosis likely: > 60 mmol/L
CFTR regulates electrical balance. Associated with chronic obstructive pulmonary disease & pancreatitis susceptibility.
Metal electrodes drive pilocarpine medicine into the skin, then sweating occurs and samples are collected in either filter paper or gauze. Sweat in the gauze can be used to figure out how much Cl- is in the sample.
Lab method for detecting HCO3-:
- Manometric - pressure changes associated with CO2 levels in patient samples.
> Natelson micro-gasometer: measure CO2 gas after acidification of serum.
> Corning 965 CO2 analyzer: measure thermal conductivity of gas produced after acidification of serum. - Spectrophotometric -
> CO2 gas released by acidification, then diffuses across silicon-rubber membrane into alkaline HCO3- buffer, CO2 converted to HCO3-, and H+ changes monitored by pH indicating dye, which changes colors. - pCO2 electrode - commonly used method to quantify CO2 gas.
> CO2 gas produced by acidification of sample, then CO2 diffuses across a silicon-rubber membrane, then pH change of bicarbonate based on electrode buffer, then pH change is monitored by glass pH electrode. The rate of change is proportional to the amount of CO2. - Enzymatic-spectrophotometric - most common method.
> All CO2 forms converted to HCO3- by addition of alkali serum.
> NADH consumption measured as decreased in 340 nm absorption.
