LAB 2 Flashcards
Absorbance spectrum
Graph of absorbance vs wavelength
Absorbance maxima
The wavelength at which the absorbance is the highest is called the wavelength maxima or the absorbance maxima
Unit for molar absorbance coefficient
Lmol-1cm-1
A 3mL corvette was filled with 1mL of solution X and 2mL of water and its absorbance at 580 nm is 0.354. Undiluted solution X has a concentration of 0.6 mmolL-1 and the path length is 1cm…what is the molar absorbance coefficient?
A=0.354
C=0.2 mmol/L (0.6 x 1 = 3 x C2… C2 = 0.2)
I= 1
0.354 = E x 0.2 x 10^-3 E= 1770 Lmol^-1cm^-1
How do we see colours…
Yellow colour reflects yellow light and absorbs all other colours for example
Heme and iron are not
Proteins
Spectrophotometry
Technique used that can typically measure the amount of visible and UV light absorbed by molecules in a solution, depending on the type of spectrophotometer used. Because different molecules absorb light at different wavelengths, we can use spectrophotometry to identify the molecules that are present in a solution.
Standard curve = pass through origin, must not go above 1.0 absorbance and cannot extrapolate (dilute instead)
Beer’s Law
States that, when monochromatic light passes through an absorbing solution, the intensity of the transmitted light decreases exponentially with increasing concentration of the absorbing solute. In other words, when taken as a log of the ration Io/I, if the concentration of a solute doubles, the absorbance of that solution doubles too.
A=ExCxl
Lambert’s law
States that, for monochromatic light transmitted through an absorbing solution, the intensity of the transmitted light decreases exponentially with increasing path length through the solution. In other words, when take as a log of the ratio Io/I, if the distance that the transmitted light has to travel through the solution doubles, the absorbance of that solution doubles too.
Because the absorbance is expressed as the log ratio of the incident light to the transmitted light, in practice this translates to…
The concentration of a solution being proportional to the absorbance of that solution (Beer’s Law) AND the intensity of the transmitted light being proportional to the path length (Lambert’s law), being related linear
Absorbance
A = Log10 x Io/I
The relationship between absorbance and concentration
When concentration increases then the absorbance also increases (proportional to each other)
The relationship between the molar absorbance coefficient and the concentration of a solution
There is no relationship - concentration and absorbance have a relationship but not the coefficient
Oxyhemoglobin and deoxyhaemoglobin
Oxygen is able to reversibly bind to the haem iron (Fe2+). When oxygen is bound, haemoglobin is called oxyhemoglobin and when oxygen is not bound it is called deoxyhaemoglobin. Haemoglobin will bind oxygen, i.e. become oxygenated when there is a lot of oxygen available in the body environment, and release the oxygen, i.e. become deoxygenate where the oxygen concentration is low.
Haemoglobin in red blood cells in the blood circulating around the body therefore becomes oxygenated in the vincinty of the lungs. COnversely, it becomes deoxygenated in the vicinity of the areas in your body where oxygen is being used in aerobic metabolism, such as your muscle
oxy has two peaks on an absorbance vs wavelength graph whereas deoxy has only one peak
Why is it usually preferable to measure the absorbance of a molecule at its absorbance maxima
Enable the best sensitivity of detection
Cooperativity and reverse oxygen binding in haemoglobin
Cooperativity for oxygen binding is observed in haemoglobin because the interaction of the oxygen molecule with one haem facilitates the binding of additional oxygen molecules to the other haem sites. Deoxyhaemoglobin is likely to be in the low oxygen affinity state (T state), but when one oxygen binds to one of the four haem groups, conformational changes in that haem group affect the structural arrangement in the adjacent globin subunits, increasing their oxygen affinity.
Whether haemoglobin binds or releases oxygen is primarily dependent on the local environment partial pressure of oxygen. High partial pressure in lung: oxygen binding, low partial pressure of oxygen at the peripheral tissues: oxygen release.
Changes in haemoglobin as it moves from the lungs to the peripheral tissues…
Lungs - tight binding of oxygen therefore want R state
Peripheral tissue - want releases therefore T state
Deoxygenation of oxyhemoglobin using live yeast
Yeast are single celled eukaryotic microorganisms that utilise oxygen and glucose to produce energy. When yeast cells are added to a oxyhemoglobin solution they will use the oxygen bound to oxyhemoglobin, deoxygenating it in the process.
OxyHb
Has Fe in the Fe2+ state, oxygenated and in the reduced form
Beer-Lambert law
The amount of energy absorbed or transmitted by a solution is proportional to the solution’s molar absorptivity and the concentration of solute. In simple terms, a more concentrated solution absorbs more light than a more dilute solution does.
