INFORME DE PONENCIA NEGATIVA PARA SEGUNDO DEBATE AL PROYECTO
V. Consideraciones de la ponente
Key Terms 154 Analytical Spectroscopy 154 Enzyme Kinetics 157 pH Buffering 164 Acid-Base Disorders 168 Anion Gap 169
Osmolarity and Osmolality 170 Osmolality Gap/Osmolarity Gap 171 Lipid Calculations 172
Creatinine Clearance 173
Learning Objectives
At the end of this chapter, the student should be able to do the following: 1. Explain and use the relationship between transmittance and absorbance 2. Explain the proportionality between absorbance and molar absorptivity,
concentration, and path length 3. Use Beer’s law properly
4. Explain when to use a standard curve, the molar absorptivity method, or the single-standard method to quantify a chromophore
5. Explain the strengths and weaknesses of end-point, two-point, and kinetic assay modes
6. Explain the significance of initial rate, KM, and Vmax
7. Relate the Michaelis-Menten equation to its plot and to its underlying model of enzyme catalysis
8. Explain the strengths and weaknesses of Lineweaver-Burk plots
9. Estimate KM and Vmax from a Michaelis-Menten plot and from a Lineweaver- Burk plot
10. Define the phenomenon of pH buffering
11. Use Ka and pKa to compare the strengths of acids
12. Using the Henderson-Hasselbalch equation, calculate the concentrations of an acid and its conjugate base necessary to prepare a buffer at a given pH
13. Use the Henderson-Hasselbalch equation to calculate any one of these quantities from the other three: pH, pKa, concentration of acid, concentra- tion of conjugate base
14. Properly apply the Henderson-Hasselbalch equation to the CO2@bicarbonate buffering system in the blood
15. Differentiate among respiratory and metabolic acidosis and alkalosis by pH, PCO2, and bicarbonate concentration
16. Calculate the anion gap, with and without potassium 17. Calculate the osmolarity of a solution from the molarity
18. Calculate the osmolarity and osmolality of plasma, given the concentra- tions of sodium, glucose, and BUN
20. Calculate the concentration of LDL cholesterol by means of the Friedewald equation 21. Calculate the creatinine clearance rate, given the required information
Key Terms
I0 I
Detector absorbance
acid dissociation constant acidosis alkalosis anion gap Beer-Lambert law buffered chromophore conjugate acid conjugate base creatinine clearance double-reciprocal plot end-point assay enzyme enzyme kinetics Friedewald equation glomerular filtration rate HDL Henderson-Hasselbalch equation hypertonic hypotonic initial rate Ka KM kinetic assay lag phase LDL linear phase Lineweaver-Burk plot lipoprotein
maximal velocity (Vmax)
metabolic acidosis/alkalosis Michaelis-Menten equation molar absorptivity
molar absorptivity method molar extinction coefficient osmolality osmolality gap osmolarity osmole osmosis osmotic pressure partial pressure pKa respiratory acidosis/alkalosis single-standard method substrate substrate-depletion phase transmittance two-point assay VLDL
AnAlyticAl SpectroScopy
Among the most important techniques in the clinical laboratory is analytical spectroscopy. It is based on the phenomenon that many chemical substances absorb light of a particular wavelength. A beam of light of known intensity (I0) is directed into a solution, and the intensity (I) of the light emerging from
the solution is then measured.
The fraction of light transmitted (I/I0) is called the transmittance (T):
T = II 0
Being a fraction, T ranges in value from 0 to 1. The light that did not pass through the sample was absorbed. For example, if T = 0.80, then 80% of the light passing through the sample was transmitted
and 20% was absorbed. Although transmittance goes down as concentration goes up, the relationship is not linear (Figure 9-1A n).
Therefore, a plot of T against concentration is more difficult to use as a standard curve than is a straight line. But the logarithm of T as a function of concentration is a straight line and, as a result, is more useful for this purpose (Figure 9-1B). Accordingly, absorbance (A) is defined as the logarithm (base 10) of the transmittance:
A K -log II
0 = -log T
Thus, if T = 0.648, then 64.8% of the light passing through the sample is transmitted and 35.2% is absorbed. The absorbance, then, or A, is
A = -log T = -log 0.648 = 0.188 Absorbance depends, logically, on the following three factors.
• The concentration of the absorbing chemical substance (the chromophore). At higher concentrations, there is more of the chromophore present to absorb the light.
• The length of the path the light takes passing through the solution. In a longer container, the light stays in contact with the chromophore for a longer period of time and, accordingly, has more opportunity to be absorbed.
• The inherent ability of the chromophore to absorb the light. This ability is quantified in the molar absorptivity or the molar extinction coefficient. For every chromophore, it is unique and constant under a given set of conditions (solvent, wavelength, temperature).
The Beer-Lambert law (also called “Beer’s law”) is the mathematical relationship among absor- bance and the three factors listed above:
A = 𝛜 : c : l Equation 1 Equation 1 Absorbance (no units) Molar absorptivity (L
#
mol-1#
cm-1) Path length (cm) Concentration (mol/L)Equation 1 is linear. As Figure 9-1B shows, absorbance is directly proportional to concentration (as it is to path length), with ϵ functioning as the proportionality constant.
n Figure 9-1 Panel A: The relationship between transmittance and
concentration is nonlinear; %T is not proportional to concentration. Panel B: Absorbance is directly proportional to concentration.
A
%T
Concentration Concentration
If A has been measured, and if the molar absorptivity and path length are known, then the concen- tration of a chromophore can be calculated by solving Equation 1 for c:
c = ϵ * lA
Suppose, for example, that we have a solution of all-trans-retinol (vitamin A) in isopropyl alcohol and we want to ascertain its concentration. In a reference book, we find that retinol in isopropyl alcohol has a molar absorptivity of 52,300 L
#
mol-1#
cm-1. If we measure the absorbance of our solution to be 0.628,and if the path length is 1 cm, then the concentration is
c = 0.628
(52,300 L
#
mol-1#
cm-1)(1 cm) = 0.000012 mol/L= 1.2 * 10-5 mol/L = 12 μmol/L
Beer’s law is especially useful when the analyte is too unstable to generate a standard curve of absor- bance versus concentration. In such a case, we calculate the concentration directly from its absorbance in the solution. This is the molar absorptivity method.
An alternative to the molar absorptivity method is the single-standard method, in which the absor- bance of only one standard solution is measured and a line is drawn through it as the standard curve. This method is useful only if we know the standard curve to be linear.
Even though absorbance is directly proportional to concentration, the relationship does not remain linear as concentration continues going up (Figure 9-2 n). For any chromophore, the concentration range in which absorbance is linear must be determined experimentally, and any absorbance reading above that range should not be trusted when used in Beer’s law.
Sometimes a chemical substance does not obey Beer’s law at any concentration, giving instead a curve across the entire range. The reasons for this behavior we leave to a chemistry textbook.
Therefore, generating a standard curve from several data points has at least two major advantages over the single-standard method.
• It can reveal nonlinearity that might be present so that (1) the sample may be diluted into the linear range or (2) the data may be fit to a curve by means of nonlinear regression.
• It averages out random errors over all the standards.
However, the additional time and cost represent one disadvantage of the standard-curve method over the single-standard method.
n Figure 9-2 Relationship between absorbance (A) and concentration
eventually becomes nonlinear.
Concentration
A
Relationship is linear. Beer’s law is VALID. Relationship is nonlinear.