Chemistry 1 - Lab Practical 1

Question 1

Identify the four basic measurement techniques seen in the modern medical laboratory.

Answer 1

Spectrometry
- Spectrophotometry
- Atomic Absorption
- Mass Spectrometry

Luminescence
- Florescence
- Chemiluminescense
- Nephelometry

Electroanalytical methods
- Electrophoresis
- Potentiometry
- Amperometry

Chromatography
- Gas
- Liquid
- Thin-layer

Question 2

Differentiate photometry and spectrophotometry.

Answer 2

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.

Question 3

Diagram the basic components of a spectrophotometer.

Answer 3

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

Question 4

Assess the function of the basic components of a spectrophotometer.

Answer 4

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).

Question 5

Examine the nature of light including how it is measured and its relationship to energy.

Answer 5

• 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)

Question 6

Given a wavelength of light, predict the color seen and absorbed.

Answer 6

< 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

Question 7

Define spectral bandpass.

Answer 7

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.

Question 8

Compare types of monochromators.

Answer 8

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

Question 9

Evaluate quality control testing on spectrophotometers to determine acceptability.

Answer 9

• 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

Question 10

Contrast absorbance and percent transmittance.

Answer 10

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.

Question 11

Convert between absorbance and percent transmittance.

Answer 11

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

Question 12

State Beer-Lambert’s Law.

Answer 12

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

Question 13

Perform calculations using Beer-Lambert’s Law.

Answer 13

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

Question 14

Predict concentration of unknown from Standard Calibration Curve.

Answer 14

• Absorbance vs. concentration
  > Results in a straight line on linear graph paper
• %Transmittance vs. concentration
  > Makes straight line on semi-log paper

Question 15

Resolve case studies involving spectrophotometry.

Answer 15

Do practice questions slide 42-47

Question 16

Describe relationships in absorbance spectrophotometry.

Answer 16

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.

Question 17

For electrolyte methodology, what is the most popular specimen used for Na+ and K+ detection?

Answer 17

• 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

Question 18

What are sources of error for Na+ and K+ measurement?

Answer 18

• 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.

Question 19

What are methods for detecting Na+ and K+?

Answer 19

1. 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.
2. 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

Question 20

Describe specimen collecting and handling for Na+ and K+.

Answer 20

• 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.

Question 21

Methods for detecting Cl-

Answer 21

Interference with other halides

1. 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.
2. 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.
3. Ion Selective Electrodes - most common method. Direct and indirect methods (same applications as with Na+/K+. Has silver (AgX) complex sensing capabilities.
4. 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.

Question 22

Specimen collection & handling for Cl-:

Answer 22

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.

Question 23

Sweat Chloride:

Answer 23

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.

Question 24

Lab method for detecting HCO3-:

Answer 24

1. 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.
2. 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.
3. 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.
4. 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.

Question 25

Facts about Lab method for detecting HCO3-:

Answer 25

Total CO2 exists as –- (H+) + (HCO3-) <> H2CO3 <> H2O + CO2
More acidic conditions support carbon as CO2, more alkaline conditions support carbon as HCO3-. At 7.4, the ideal pH, most carbon is H2CO3, but this never happens.
> dissolved CO2 (3%)
> carbamino derivatives of plasma protein (33%)
> bicarbonate (64%)
Normal values: healthy adult 22-30 mmol/L (method dependent & physiological adjustment dependent (i.e. exercise)).
If given CO2 values, then it is just bicarbonate values. If given HCO3- values, then it is similar to total CO2.

Question 26

Describe the Enzymatic-spectrophotometric method for HCO3-:

Answer 26

> All CO2 forms converted to HCO3- by addition of alkali serum.
> NADH consumption measured as decreased in 340 nm absorption.
Phosphoenolpyruvate + (HCO3-) + phosphoenolpyruvate carboxylase > oxaloacetate
(Substrate) (Patient sample) (Enzyme)
Oxaloacetate + NADH + Malate dehydrogenase > Malate + (NAD+)

Question 27

Specimen collection and handling for HCO3-:

Answer 27

Serum, heparinized plasma
Cannot use other anticoagulants (disturb erythrocyte and plasma CO2). Whole blood cannot be used due to heme-bound CO2 and carbamino-bound CO2.
Major error associated with total CO2 measurement occur with handling of sample: exposure to air should be minimal and centrifugation at 37C. Tightly stoppered before analysis.
Stability: serum or plasma total CO2 is stable for several days at 4C.

Question 28

Buffers of the body:

Answer 28

Bicarbonate: immediate buttering capacity
Hemoglobin: major blood buffering capacity
Phosphorous: (H2PO4- > HPO4^2-) small contribution - important in urine
Albumin protein: little contribution
Normal pH 7.35-7.45
> Derivation of the Henderson-Hasselbach equation

Question 29

Key points of Henderson-Hasselbach equation:

Answer 29

pH = pK + log([HCO3-]/apCO2)
pK = 6.11 / a = 0.0301 mmol/L/mmHg
[HCO3-] cannot be accurately measured, but can be calculated from TCO2 & pCO2 values, which can be measured in a clinical setting.
TCO2 = dissolve CO2 + [HCO3-] + [H2CO3]
> In the body [H2CO3] &laquo_space;dissolved CO2 & [HCO3-]
> So TCO2 = dissolve CO2 + [HCO3-]
> Dissolved CO2 = a(pCO2), where a is the Brunson’s constant
> [HCO3-] = TCO2 - a(pCO2)

Question 30

Oxygen Saturation Curve:

Answer 30

Shift to right (decreased affinity): up pCO2, down pH, up temperature, up 2,3 DPG (exercise b/c increased metabolism/metabolic rate).
> when pCO2 goes up, pH does down (hypoxia).
> 2,3 DPG = diphosphoglycerate
Shift to left (increased affinity): down pCO2, up pH, down temperature, down 2,3 DPG.

Question 31

Acid-Base Disorders:

Answer 31

Metabolic acidosis: lower pH, lower [HCO3-], normal pCO2
Respiratory acidosis: lower pH, normal [HCO3-], up pCO2
Metabolic alkalosis: up pH, up [HCO3-], normal pCO2
Respiratory alkalosis: up pH, normal [HCO3-], lower pCO2
> When the pH changes in the blood, it is sensed by the nervous system (hypothalamus). Then the body tries to make compensations to correct the pH.
> 1st compensation (immediate compensation) = respiratory compensation. An increase in respiratory rate will help with metabolic acidosis + metabolic alkalosis.
> If you have respiratory acidosis or respiratory alkalosis, then the respiratory system is damaged and won’t use respiratory compensation. It will instead use metabolic compensation.
> Long-term compensation: happens when the patient’s underlying condition is fixed, then it moves to long-term compensations. In respiratory conditions, compensations are both metabolic and respiratory as respiratory condition has been fixed.

Question 32

Base deficit disorders - Metabolic Acidosis:

Answer 32

Two forms:
Increased organic acids → decrease [HCO3-]
Decreased [HCO3-] due to diarrhea
> both decrease HCO3- in the HH equation. When HCO3- decreases, then the log value gets smaller, so pH decreases, ([HCO3-]/apCO2) < 20
Causes:
Uncontrolled diabetes
Fasting or fad diets
Strenuous exercise
Hypoxia
Renal tubular acidosis
Liver disease
Ingestion of salicylic acid or other poisons
Physiological Response:
> Compensated metabolic acidosis: initially as acidemia occurs the body compensates by hyperventilating. Increases pCO2 in the HH equation here ([HCO3-]/apCO2).
> Over the long term the kidney will excrete organic acids and exchange H+ for Na+ (only if the underlying cause of acidosis is corrected).
Laboratory findings:
Increased lactic acid
Increased anion gap
Decreased pH
Decreased [HCO3-]
Decreased O2 saturation
Decreased pCO2
Increased K+
Treatment: correct underlying cause. In case of diabetes → insulin. Then add HCO3-.

Question 33

Base deficit disorders - Respiratory Acidosis:

Answer 33

Ratio is decreased ([HCO3-]/apCO2) < 20
Causes:
> Pulmonary edema
> Bronchial constriction
> Pneumonia
> Asthma
> Emphysema
> Apnea - stop breathing
> Bradycardia - slow heart beat
> Respiratory depression
> Respiratory distress syndrome (RDS)
Physiological response:
Compensated Respiratory Acidosis: development of metabolic alkalosis, body compensates by retaining Na+ and HCO3- and hyperventilating - development of normal pH, but with excessive HCO3-.
Over the long term, the kidney will excrete organic acids and exchange H+ for Na+ only if the underlying cause of acidosis is corrected.
Laboratory findings:
Increased pCO2, with renal compensation (increased HCO3-)
Treatment: treat underlying disorder (initial treatment may also require Na-HCO3-).

Question 34

Base excess disorder - Metabolic Alkalosis:

Answer 34

([HCO3-]/apCO2) > 20, via increased HCO3-
Causes:
Hypochloremic (decreased Cl-) alkalosis (prolonged vomiting, increased renal reabsorption of HCO3-).
Hyperaldosteronism (Cushing’s Syndrome) may cause hypokalemia (increased [HCO3-] in plasma as K+ moves from inside to outside the cells with H+ moving in).
Physiological response:
Compensated metabolic alkalosis: body compensates by slowing respiratory system, thus increasing pCO2.
Over the long term the kidney will excrete express HCO3-, if underlying condition is corrected.
Laboratory findings:
Ratio ([HCO3-]/apCO2) is increased, but so does pCO2 (compensation).
Urine will contain titratable HCO3-.
Treatment: administer NaCl or KCl (depending on hypokalemia), NH4Cl if alkalosis is severe.

Question 35

Base express disorder - Respiratory Alkalosis:

Answer 35

([HCO3-]/apCO2) > 20, decreased apCO2.
Causes:
> Hyperventilation (excessive crying, CNS control of respiration, asthma, fever, pulmonary embolism).
Physiological response: compensated respiratory alkalosis: kidney will excrete excess HCO3-.
Laboratory findings:
> Hyperventilation leads to decreased pCO2 (a cause of Respiratory alkalosis).
> Increased amounts of HCO3- in urine
Treatment: sedatives, have patients breath in paper bags.

Question 36

Specimen of blood-gas analysis:

Answer 36

Whole blood (unclotted, unseparated)
> Collect arterial, venous, “capillary”
> Arterial: most common
> Venous: fine for acid-base
> “Capillary” can be considered as “arterialised” if limb is warmed to 45*C (increases blood flow) prior to collection.

Question 37

Preanalytical Considerations of blood-gas analysis:

Answer 37

Specimen container:
> Whole blood is collected in glass or plastic syringes or capillary tubes.
> Glass is inert (best choice for pO2), well stoppered, gas-tight for 2 hours.
> Plastic is permeable to gas (ok for pH, pCO2, HCO3-, base excess and electrolytes). Plastic containers are not ideal, but if used for pO2 samples, the sample must be analyzed within 15 minutes of being drawn.
Anticoagulants:
> Heparin
> Want to use the minimum amount necessary, otherwise decreased pH, [HCO3-], base excess, and Ca2+.
> Glass containers requires slightly higher amounts of heparin than plastic.
> 2 forms of heparin:
Dry (advantage: little dilution of specimen, disadvantage: takes time for heparin to dissolve and mix).
Liquid/solution (advantage: fast mixing, disadvantage: dilution of sample). However, solution anticoagulants can also decrease electrolytes, HCO3-, Hgb, and increase pO2 because O2 in heparin solution is equilibrated with atmospheric air.
Technique of collection:
> Status of patient - calm, steady ventilation for 15 minutes, 30 minutes for patients receiving artificial air or CO2. Pain/anxiety should be minimalized.
> Sample should be drawn anaerobically in presence of anticoagulants. Samples with air bubbles should be discarded.
> Keep hemolysis to a minimum.
> Venous occlusion by tourniquet < 2 minutes.
Storage and transport:
> Temperature & time are critical
> Temperature - specimens in glass should be placed on ice/water slurry immediately. Specimens in plastic should be analyzed immediately.
> Time - stability in glass about 2 hours & plastic 15 minutes. Prolonged storage may decrease Ca2+ & increase K+ (recommended that samples be analyzed within 1 hour).

Question 38

pH Electrode:

Answer 38

Calibration: with buffer solutions that bracket pH of sample
Calibrators, buffers, and electrodes must all be at the same temperature.

Question 39

pCO2 Electrode:

Answer 39

Electrode takes advantage that pH has a linear relationship to log pCO2 over a range of 10-90 mmHg (normal pCO2 is 35-45 mmHg).
Invasive (blood drawn). Calibration is zero gas and a slope gas (40 mmHg) - usually bubbled through a humidifier to include contribution of water vapor pressure.
Non-invasive (transcutaneous pCO2 (tcpCO2). Same detection principle as pCO2 electrode above except detection occurs on skin (requires skin to be warmed to 42-43*C).
Electrode collects for CO2 accumulation during analysis.
> Calibration is 2 gas mixture (same as above)

Question 40

pO2 Electrode:

Answer 40

Specificity of electrode dependent on membrane permeability to dissolved O2.
O2 is consumed. Current is proportional to [O2].
Reference electrode generates constant ~600 mv e-.
Invasive: requires blood to be drawn
> Normal pO2 ~80-110 mmHg
> Calibration: 2 gasses (zero and another with O2), most instruments are self-calibrating.
Non-invasive: Transcutaneous pO2 (tcpO2) - same detection principle as pO2 electrode, except detection occurs on skin (requires skin to be warmed to 42-43*C).
> Calibration is atmospheric O2.