Spectrophotometry

A number of determinations in biochemistry make use of the fact that many substances absorb electromagnetic radiation in the visible or ultraviolet part of the spectrum, less often infrared radiation. The extent to which a substance absorbs light of different wavelengths (ie, the absorption spectrum) depends on the structure of the compound. The amount of light of a certain wavelength that is absorbed by, for example, a substance dissolved in a solution depends on the concentration of the substance. The measurement of light absorption by a sample is among the most widely used techniques in biochemistry and is referred to as photometry (when measured at one or more specific wavelengths) and spectrophotometry(if measured over a certain continuous range of light wavelengths).

Colour of fabrics
A number of substances contain a valence electron that can be excited to a higher energy level by electromagnetic radiation. Such a substance then absorbs radiation of a certain wavelength with photon energy corresponding to the energy difference of the two electron levels. If the absorbed radiation lies in the visible part of the spectrum, the substance will appear colored to the human eye (it will have a color complementary to the color of the absorbed light). Unfortunately, it is not easy to predict the color of a substance based on its chemical structure, nor is it possible to unambiguously infer the composition of the substance based on the absorption spectrum. However, from our point of view, three groups of substances that are often colored are significant:


 * 1) Substances containing a system of conjugated double bonds, the molecule of which is not symmetrical. If we imagine a symmetric conjugated double bond system, it can exist in two resonance states that are energetically equivalent: The presence of an asymmetric substituent causes the energies of the two states to differ. The energy difference often corresponds to the energy of a photon from the visible part of the spectrum. Typical representatives can be dyes with a polymethine chain (–CH=CH–CH=CH–) or azo dyes (–N=N–). Substances with aromatic or heterocyclic structures bound to a common central atom (e.g. triphenylmethane dyes) behave similarly.
 * 2) Also, the d and f valence electrons often determine the color of the compound. They tend to be present in coordination covalent bonds of complex compounds . For example, anhydrous copper sulfate CuSO 4 is colorless, while its pentahydrate CuSO 4 •5H 2 O and its aqueous solution are blue: in both cases, copper enters a complex with water [Cu(H 2 O) 4 ] 2+ . In a similar way, colored and complex compounds of other transition metals (Fe, Cu, Cr, Mn, Ni, Co) tend to be complex-bound metal also in the colored proteins of hemoglobin and cytochromes.
 * 3) Ions that contain a transition metal with a high oxidation number as a central atom, e.g. MnO4-, Cr2O7 2-, are also colored.

Analytical methods used in medicinal chemistry and biochemistry make use of all three groups of colored compounds. Systems of conjugated double bonds are often formed in reactions in which the analyte condenses with a suitable chromogen (e.g. creatinine with picric acid in the Jaffé reaction, diazo coupling reactions in the detection of bilirubin ), or are formed by oxidation of a chromogen that contains one less double bond (oxidation of benzidine derivatives in peroxidase reactions). The formation of colored complexes is used, for example, in the determination of proteins by the so-called biuret reaction (Cu 2+ complexes with O and N peptide bonds) or in the detection of a number of substances, e.g. with FeCl 3. Color changes during the reduction of Cr 6+ to Cr 3+is used, for example, in the detection of ethanol in exhaled air.

Absorption of monochromatic light can also be conditioned by events other than electron excitation. These are primarily changes in the various oscillation energies of atoms in molecules and the rotational energies of entire molecules. These principles are used more in fluorimetry. From the point of view of medical biochemistry, they are much less important than the above principles.

Transmittance
The amount of light of a certain wavelength that passed through the sample is described by the transmittance (lat. transmitto, I convert, let through). It is defined:

$$T = \frac{I}{I_0}$$

where
 * T is the transmittance,
 * I is the intensity of light that passed through the sample,
 * I 0 is the intensity of the light that entered the sample.
 * I 0 is the intensity of the light that entered the sample.

V praxi by bylo nevhodné měřit přesně obě intenzity: kromě vlastností vzorku jsou ovlivněny i absorpcí a odrazem světla na stěnách kyvety a v optice fotometru, prostředím, v němž probíhá měření atd. Proto se obvykle měří transmitance relativně vzhledem ke slepému vzorku. Nejprve se změří intenzita světla procházejícího slepým vzorkem (blankem, referenčním vzorkem), tj. roztokem obsahujícím všechny složky vyjma stanovované barevné látky. Pak se za stejných podmínek měří intenzita světla procházejícího neznámým vzorkem. Transmitance je pak definována vztahem

$$T = \frac{I_v}{I_b} $$

kde
 * T je transmitance,
 * Iv je intenzita světla, které prošlo vzorkem,
 * Ib je intenzita světla, které prošlo slepým vzorkem.

Měří-li se transmitance tímto způsobem, není třeba se zabývat nespecifickými ztrátami intenzity světla. Intenzita světla, které prochází slepým vzorkem, se považuje za 100 % (tj. transmitance blanku je 100 %) a transmitance vzorků absorbujících světlo dané vlnové délky je vždy menší než 100 %.

Transmitance roztoku, který obsahuje barevnou látku, záleží na
 * vlastnostech absorbující látky,
 * vlnové délce procházejícího světla,
 * množství absorbující látky, tj. na její koncentraci v roztoku a na tloušťce kyvety.

August Beer (1825–1863) poprvé formuloval závislost transmitance na těchto veličinách matematicky. Za předpokladu, že se použije monochromatické světlo, platí

$$T = 10^{-\epsilon \cdot l \cdot c}\,\!$$

kde
 * T je transmitance,
 * &epsilon; je molární dekadický absorpční koeficient (konstanta specifická pro danou látku při určité vlnové délce),
 * l je optická délka kyvety,
 * c je látková koncentrace absorbující látky.

Algebraickými úpravami můžeme transmitanci vyjádřit také

$$\log T = -\epsilon \cdot l \cdot c\,\!$$ nebo $$-\log T = \epsilon \cdot l \cdot c\,\!$$,

na základě čehož se definuje absorbance a optická hustota

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