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CHMI 2227E Biochemistry I. Refresher: acid-base chemistry spectrophotometry. Acid base chemistry - strong acids. HCl H + + Cl -. pH = - log [H + ]. Where [H + ] is in molar (M) concentration units. Strong acids dissociate completely in water. HA H + + A -. Ka = 1
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CHMI 2227EBiochemistry I Refresher: acid-base chemistry spectrophotometry CHMI 2227 - E.R. Gauthier, Ph.D.
Acid base chemistry- strong acids HCl H+ + Cl- pH = - log [H+] Where [H+] is in molar (M) concentration units. • Strong acids dissociate completely in water. CHMI 2227 - E.R. Gauthier, Ph.D.
HA H+ + A- Ka = 1 10pKa Acid base chemistry- weak acids • Where: • HA is the undissociated acid • A- is the conjugated base • of acid HA • Weak acids do not dissociate completely in water; • The extent to which the weak acid will dissociate is indicated by the dissociation constant (Ka). Ka = [H+] x [A-] [HA] pKa = -log Ka CHMI 2227 - E.R. Gauthier, Ph.D.
Acid base chemistry- weak acids • The pH of weak acids solutions can be determined by first calculating the [H+], taking in consideration the Ka; Ka = [H+] x [A-] [HA] pH = - log [H+] • Alternatively, one can also use the Henderson-Hasselbach equation: pH = pKa + log [A-] [HA] CHMI 2227 - E.R. Gauthier, Ph.D.
HA H+ + A- pH 100% A- • Mid-equivalence point: • 50% HA • 50% A- pKa pH at mid-equivalence point = pKa 100% HA 0.5 NaOH Acid base chemistry- weak acids: titration • Adding a strong base to a weak acid solution will progressively convert more and more HA to A-. • Notice that the pH doesn’t change significantly near the pKa: the solution is said to be buffered. CHMI 2227 - E.R. Gauthier, Ph.D.
CH3-COOH CH3-COO- + H+ Acid base chemistry- weak acids • Example 1: What is the pH of a 0.1 M solution of acetic acid (Ka = 1.76 x 10-5M). Ka = 1.76 x 10-5 M = [H+] x [A-] = [H+]2 [HA] [HA] [H+]2 = 1.76 x 10-5 M x [HA] = 1.76 x 10-5 M x 0.1M [H+] = 1.33 x 10-3 M And finally: pH = -log [H+] = 2.88 CHMI 2227 - E.R. Gauthier, Ph.D.
CH3-COOH CH3-COO- + H+ Acid base chemistry- weak acids • Example 2: What will be the pH of a solution made of 0.3 M acetic acid and 0.1 M acetate (pKa = 4.8). pH = pKa + log [A-] [HA] pH = 4.8 + log 0.1M 0.3M pH = 4.8 + (-0.477) pH = 4.3 CHMI 2227 - E.R. Gauthier, Ph.D.
Spectrophotometry Cuvette Light source Detector Intensity of transmitted light same as incident light Transmitted light Incident light Intensity of transmitted light less than incident light Transmitted light Incident light In other words, the blue solution absorbed some of the incident light. CHMI 2227 - E.R. Gauthier, Ph.D.
Different light sources can be used. In biochemistry, the two most widely used light sources are: Visible light (for coloured compounds); Ultraviolet light: uncolored compounds with aromatic rings/conjugated double bonds. For this reason, we call this method UV-Vis spectrophotometry. Spectrophotometry CHMI 2227 - E.R. Gauthier, Ph.D.
The wavelength to use depends on the type of compound you’re interested in. Usually, preliminary experiments have to be performed to find the wavelength where your compound will absorb the most; increases sensitivity of the assay. Absorbance intensity 400 450 500 550 600 Wavelength (nm) Spectrophotometry CHMI 2227 - E.R. Gauthier, Ph.D.
The big deal with spectrophotometry is that is allows you to use the amount of absorbed light to measure things. We use absorbance instead of transmittance because it’s easier to see differences in values. For example: Solution A: 1M: 65% transmittance and 0.25 absorbance 2M: 70% transmittance and 0.5 absorbance The relationship between the transmittance and absorbance is given by the Beer-Lambert equation. Spectrophotometry CHMI 2227 - E.R. Gauthier, Ph.D.
Spectrophotometry- Beer-Lambert equation Where: T = transmittance A = absorbance l = light path (cuvette size)(cm) c= concentration of analyte (M) e = absorption coefficient (M-1cm-1) • Usually, l = 1 cm. • e is a property of the molecule studied under standardized conditions, and is found in handbooks. • So: if you know A, l and e, you can immediately know c. • However, e is rarely known, or not valid under the conditions used in the lab. • WHAT TO DO?????????????? -logT = log (1/T) = ecl =A CHMI 2227 - E.R. Gauthier, Ph.D.
Very frequently used in biochemistry; Pretty simple: The absorbance of a set of solutions of the compound of interest, of known concentrations, is first determined. A graph of the absorbance vs concentration is then made. THIS IS YOUR STANDARD CURVE. The absorbance of the same compound, but of unknown concentration, is then determined. As long as the absorbance of the unknown fits in the linear part of the standard curve, you can determine the concentration of your sample. Non-linear part of the curve. BEWARE!!! Absorbance of the unknown Absorbance intensity Concentration of the unknown 3 6 9 12 Concentration(units) Standard curves CHMI 2227 - E.R. Gauthier, Ph.D.
Enzymatic reactions: The formation/exhaustion of a light-absorbing molecule as a function of time can be determined by spectrophotometry. This allows us to monitor the progress of enzymatic reactions. Sample detection: Proteins absorb at 280 nm; UV-Vis is often used to follow the progress of protein purification by monitoring the absorbance at 280 nm. Sample purity: Pure DNA samples absorb at 260 nm and 280 nm in such a way that the ratio of the absorbance A260/A280 is around 2. A A260/A280 ratio of less than 2 tells you that your DNA is contaminated with proteins. Spectrophotometry • Spectrophotometry is used all the time in biochemistry, and not only to measure the concentration of molecules: CHMI 2227 - E.R. Gauthier, Ph.D.
