LAB 2

Question 1

Absorbance spectrum

Answer 1

Graph of absorbance vs wavelength

Question 2

Absorbance maxima

Answer 2

The wavelength at which the absorbance is the highest is called the wavelength maxima or the absorbance maxima

Question 3

Unit for molar absorbance coefficient

Answer 3

Lmol-1cm-1

Question 4

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?

Answer 4

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

Question 5

How do we see colours…

Answer 5

Yellow colour reflects yellow light and absorbs all other colours for example

Question 6

Heme and iron are not

Answer 6

Proteins

Question 7

Spectrophotometry

Answer 7

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)

Question 8

Beer’s Law

Answer 8

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

Question 9

Lambert’s law

Answer 9

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.

Question 10

Because the absorbance is expressed as the log ratio of the incident light to the transmitted light, in practice this translates to…

Answer 10

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

Question 11

Absorbance

Answer 11

A = Log10 x Io/I

Question 12

The relationship between absorbance and concentration

Answer 12

When concentration increases then the absorbance also increases (proportional to each other)

Question 13

The relationship between the molar absorbance coefficient and the concentration of a solution

Answer 13

There is no relationship - concentration and absorbance have a relationship but not the coefficient

Question 14

Oxyhemoglobin and deoxyhaemoglobin

Answer 14

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

Question 15

Why is it usually preferable to measure the absorbance of a molecule at its absorbance maxima

Answer 15

Enable the best sensitivity of detection

Question 16

Cooperativity and reverse oxygen binding in haemoglobin

Answer 16

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.

Question 17

Changes in haemoglobin as it moves from the lungs to the peripheral tissues…

Answer 17

Lungs - tight binding of oxygen therefore want R state

Peripheral tissue - want releases therefore T state

Question 18

Deoxygenation of oxyhemoglobin using live yeast

Answer 18

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.

Question 19

OxyHb

Answer 19

Has Fe in the Fe2+ state, oxygenated and in the reduced form

Question 20

Beer-Lambert law

Answer 20

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.