Biochemists frequently use UV spectroscopy to study conformational changes in proteins - how they change shape in response to different conditions. When a protein undergoes a conformational shift (partial unfolding, for example), the resulting change in the environment around an aromatic amino acid chromophore can cause its UV spectrum to be altered.

Detection of Impurities:-

                   UV rays are best method for detecting impurities in organic compounds. Thin layer chromatography is used for detection of impurities. A small spot is kept on the TLC plate which is then allowed to run in appropriate solvent media. Different compounds travel different distance based on their polarity which can be visualized on the TLC plate under a UV lamp. Extra spots on TLC plate can be observed due to impurities in the sample and it can be compared with the spot standard. This is qualitative analysis.

UV Detector in HPLC:-

HPLC is a high quality purification technique. It stands for high performance liquid chromatography formally high pressure. This technique is based on following explanation: UV detectors are usually of variable wavelength and can be used to detect the molecules with absorption maxima above 210nm by measuring the absorbance of the eluent. It provides a very fast detection and separation of a reaction mixture. When the compound is eluted from the HPLC column, it absorbs UV radiation at the appropriate wavelength. The amount of UV radiation absorbed is directly proportional to the amount of a particular compound that is passing through the column at that time.

Determination of concentration of the compounds:-

It is quantitative determination of compounds by the absorbance of particular It UV rays. The equation is given below to determine concentration of compounds:

 -logT = ℇbc

 

                  Where, £ is extinction coefficient, c is concentration, b is the length of the cell that is used in UV spectrophotometer. Io is the intensity of the incident radiation. I intensity of transmitted radiation. Lambert Beer law can have explained by estimation of mixture of anthracene and naphthalene these compounds have a lot of conjugated double bonds in ring structure thus they absorb in the UV region. The UV spectrum of an ethanol exertion of anthracene is shows detector a lambda max 375nm whereas naphthalene does not absorb in this region in mixture we can calculate amount of anthracene by lam. We can measure absorption of mixture at 375nm and by using equation concentration of pure anthracene can be calculated. In analytical industry concentration of vitamin A1 and vitamin A2 in natural fats or oils can be estimated by measurement of the intensity of peaks at 325nm and 351nm by comparing the intensities of solution of these vitamins with the intensities of known solution of these two enable us to calculate the concentration of A1 and A2. Both vitamins are conjugated double bonded structures. Estimation of Argo sterol in fats, anthracene in benzene, carbondisulphide in carbon tetrachloride and chlorophyll in plant material can be estimated by Beer law.

 

Stereo-chemical Studies:-

The UV rays provides a useful tool for studying stereoisomerism in certain molecules. In geometrical isomers the trans isomer exhibits absorption maximum at a longer wavelength than the cis-stilbene absorbs at 283nm and trans-stilbene at 290nm. Cisstilbene has distortion.

 

Determination of Metal Halide Complexes and Lanthanides:-

The halide complexes of metals are the main inorganic substances absorbing UV radiation. Tellurium is determined as its iodide complex [TeI6]2-, by measurements at 335nm. Characteristics intense absorption bands of lanthanides appear in UV region.

 

TautomericEquilibria:-

In many tautomeric systems, one form has a more highly conjugated chromophore and hence stronger UV absorption than the other. For example, in ethyl acetoacetate the keto form has only the weak n→π bond of an isolated carbonyl group. Consequently, a measure of the properties of the tautomer at equilibrium in various solvents can be obtained from the strength of the 244nm bond. For example, 2-hydroxy pyridine and 2-pyridone exist in tautomeric form latter preferred and equilibrium shifts toward 2-pyridone because it is alpha beta unsaturated carbonyl ketone.

 

Determination of Ionization Constants:-

There are many organic compounds which are found more than one form. The PH value of the system decides as to which form would exist. The ionization constants of the compounds can be determined spectrophotometric ally.                                                                                   

 

Determination of amount of enzymes:-

Enzymes are proteins present in living tissue. Enzymes cannot measure directly because their action is being the catalysing of the various reactions in the body but their catalytic properties allow their estimation from the speed of the reactions which they catalyse in the body. Enzymes have many uses as reagents or as labels that can be attached to the other molecules to permit their indirect detection and measurement. It has the widest use in the field of clinical diagnosis is as an indicator of tissue damage when cells are damaged by disease enzymes leak into the bloodstream and the amount present indicates the severity of the tissue damage now indirectly we can measure the amount of enzyme present using UV spectroscopy. Now relative proportional of different enzymes in the body can be used to diagnose of diseases of liver pancreas or other organs. For example, reduction of NAD (nicotinamide adenine dinucleotide) to NADH. Spectrum of NAD and NADH can plot by taking absorbance on y axis and wavelength on x axis. If absorbance is measured at 340nm the readings will increase as the reaction progresses to the formation of NADH. Another example is the protonation of pyruvate anion in the presence of NADH and an enzyme lactate dehydrogenase to lactate and NAD+. NADH is the only species which can absorbs light at 340nm, by measuring the decrease in the absorbance at 340nm the rate of reaction can determine and its kinetics can study. A graph is plotted b/w absorbance at 340nm versus time on x axis a cure is obtained showing decrease in absorbance with progress of time.

Dissociation constants of acids and bases:-

The pka of compound can measure. Compound absorbs UV light e.g. phenoxide ion has a lambda max at 287nm as it has increased conjugation if absorbance at 287nm is determined as a function of pH the pka of phenol can be noted by Henderson equation: pH= pka+ log(A-)/(HA) plotting a graph b/w the absorbance versus the wavelength at different pH values the ratio of the concentration of the anion compared to the acid or divided by the acid can determined and hence the pka value of the compounds can be calculated.

 

Distinction between conjugated and non-conjugated compounds:-

                                      (CH3)2C=CH—CO—CH3                                      (1)

                                      CH2=C(CH3)—CH2—CO—CH3                      (2)

End to 2 pi star band for the carbonyl group as in structure 1 appear in longer wavelength as it is alpha beta unsaturated ketone. It is conjugated, the carbonyl is conjugated with the double bond present. However, in comparison to the absence of conjugation in compound 2 .double bond is separated by methylene group thus the conjugation is broken does the same band appears at a shorter wavelength.

 

Differentiation between equatorial and axial confirmations:-

The end to pi star transition which comprises of the band appears at a longer wavelength in alpha, beta unsaturated ketones and is strongly influenced by the presence of a polar group in in the gamma position now it has been noted that the effect of an axial substituent to displace band to a longer wavelength is greater as compared to that observed in its equatorial isomer.

 

Study of charge transfer complexes:-

The formation of charge transfer complex occurs b/w molecules which when mixed allow the transfer of electronic charge space from an electron rich centre to an electron deficient centre with molecular orbitals having suitable energy and symmetry to do the same. The filled pi orbitals in the donor molecule overlaps with the depleted orbital in the acceptor molecule and generate two new molecular orbitals thus transition b/w these newly formed orbitals are responsible for the new absorption bands which are observed in the charge transfer complexes, the two new molecular orbitals formed. Donor and acceptor orbitals combine to form two new orbitals (a and b). a is lower in position than b. Donor is HOMO (highest occupied molecular orbital) and acceptor is LUMO (lowest unoccupied molecular orbital) combine to give charge transfer complex MO. New electronic transitions of long wavelength are then possible b/w a and b. For example, iodine (donor of charge transfer) and benzene brown complex has a lambda max at 290nm. Similarly, in the aniline(acceptor) and tetracyano ethylene(donor) deep blue complex has a lambda max at 600nm while, aniline has 280nm and tetracyano ethylene has lambda max 300nm. Now brown colour of iodine in benzene or the appearance of deep blue colour when tetracyano ethylene is added to a chloroform solution of aniline may be explained due to the formation of electron charge transfer complex. The structure of most charge transfer complexes can be visualized as a face to face association on a one is to one donor is to acceptor basis to provide maximum overlap of pi orbitals of benzene ring. Now the lambda max of benzene is 255nm while for iodine in hexane is 500nm. The charge transfer complex that is benzene and iodine displays an intense additional band at 290nm.

 

Forensics:-

UV is an investigative tool at the crime scene helpful in locating and identifying bodily fluids such as semen, blood and saliva e.g. ejaculated fluids or saliva can be detected by high power UV sources, irrespective of the structure or colour of the surface the fluid is deposited upon. UV-V is micro spectroscopy is also used to analyse trace evidence, such as textile fibres and paint chips as well as questioned documents.

Enhancing contrast of inks:-

Using multi-spectral imaging it is possible to read an illegible papyrus, such as the burned papyri of the villa of the papyri or of oxyrhynchus or the Archimedes palimpsest. The technique involves taking pictures of the illegible document using different filters in the ultraviolet range, finely tuned to capture certain wavelengths of light. Thus the optimum spectral portion can be figural for distinguishing in V from paper on the papyrus surface. Simple NOV sources can be used in K on vellum.  

Sanitary compliance:-

Ultraviolet light helps detect organic materials deposits that remain on surfaces where periodic cleaning and sanitizing may have failed. It is used in the hotel industry, manufacturing and other industries where levels of cleanliness or contamination are inspected.

 

UV-VISIBLE SPECTROSCOPY FOR CLINICAL ANDPRE-CLINICAL APPLICATION IN CANCER:-
 

Methods of optical spectroscopy which provide quantitative,physically or physiologically meaningful measures of tissueproperties are an attractive tool for the study, diagnosis,prognosis, and treatment of various cancers. Recentdevelopment of methodologies to convert measuredreflectance and fluorescence spectra from tissue to cancerrelevant parameters such as vascular volume, oxygenation,extracellular matrix extent, metabolic redox states, andcellular proliferation have significantly advanced the field oftissue optical spectroscopy.
 

INTRODUCTION:-
 

There is a great need to accurately quantify predictivebiomarkers in vivo for the diagnosis, prognosis and treatmentof cancers. Since current approaches in cancer managementare generic across patients and involve empirical routinesthere is a growing emphasis toward developing individualizedand personalized approaches which are based on detection ofmolecular, metabolic and physiological biomarkers.Traditional biomarkers include features such as the tumorgrade, size and/or the number of local lymph nodes withmetastasis.
 

BIOMARKERS OF CANCER:-

Vascular and metabolic factors:
Oxygenation and hypoxia:-

Oxygenation, particularly, the lack of it, is widely recognized as acrucial factor that influences the growth rate, metabolism, treatmentresistance and metastatic behavior of cancer cells . Hypoxicmicroenvironments have routinely been identified in solid tumors ofalmost all tissues. Numerous studies have investigated the linkbetween clinical outcomes and hypoxia using a variety of differentmethods to date . All of these studies have demonstrated that
hypoxia is clearly related to clinical outcome, which motivates theimportance of measuring it in vivo.
Currently, methods to measure tumor hypoxia can be divided intotwo classes, indirect and direct. The gold standard of direct tissuehypoxia measurement is through electrode polarography.
 

ANGIOGENESIS AND BLOOD VOLUME:-

Irregular vasculature has previously been identified has a hallmarkof cancer . Given that tumor cells have a constant need for newblood vessels to nourish their growth, solid tumors persistentlysprout new segments of vessels to the existing vascular systemleading to a highly irregular, leaky and chaotic network of bloodvessels . The sprouting of new vessels is facilitated by overexpression of the vascular endothelial growth factor (VEGF), which isknown to be upregulated under hypoxic conditions . This increasedtumor vascularization eventually paves the way for a small, localizedtumor to become an enlarged mass and subsequently metastasize toother distant sites.
 

REDUCTION-OXIDATION STATE OF THECELL:-

Cellular respiration occurs via the electron transport chain in all aerobiccells and in the mitochondrial membranes of these cells, reactive oxygenspecies (ROS) are generated during oxidative phosphorylation. There areseveral complex cellular biochemical pathways that help cells protectthemselves against low levels of ROS and free radicals by forming anetwork of redox buffers (which include theNAD(P)H/NAD(P)+ species). these mechanisms might be rendereddysfunctional under abnormally high levels of ROS leading to oxidativestress in the tumor microenvironment. onset of hypoxia within solidtumors causes the cells to prefer anaerobic glycolytic pathways overaerobic oxidative phosphorylation to meet their energy needs, which inturn influences both the amount of ROS produced, and the amount ofNADH/NAD+ redox buffer available in these cells. There is currently noaccepted clinical gold-standard to estimate the redox status of tumors,though detection in tumors has been achieved via the use of.

 

MORPHOLOGICAL FACTORS:-

There are significant changes in cellular morphology andstructure that are associated with the onset and progressionof cancer. Pathologists routinely use microscopic differencesobserved in cellular and nuclear features including shape,size, crowding, chromatin organization and DNA structure inbiopsied tissues to diagnose, prognosticate and stagedisease.
 

OPTICAL SPECTROSCOPY:-
 

Methods of optical science and engineering have been developed forcancer detection and diagnosis and more recently to assess responseto therapy in a variety of tissue sites for applications in both preclinical and clinical studies . The interaction of light with complex
media such as biological tissues, is characterized by processes thatdepend on the physical nature of the light and the specific tissuemorphology and composition . The incident light can be scattered(elastically or inelastically) multiple times due to microscopicdifferences in the index of refraction of cells and subcellularorganelles within the tissues, and may be non-radiatively absorbed bychromophores present in the medium or by fluorophores, whichrelease their excess energy by radiative decay, producingfluorescence. The remitted fluorescent light can, in turn, be multiplyscattered or absorbed. Although complex, these optical responsescan be measured by a variety of spectroscopic techniques andprocessed through rigorous computational or theoretical models to
 

In optical spectroscopy, the wavelengths of illumination spanthe ultraviolet (UV) through the near-infrared (NIR) wavelengths.In steady-state reflectance spectroscopy, a broadband light
source is used for illumination and a spectrum of the reflectedlight is collected , while in steady-state fluorescencespectroscopy a narrow spectral-band of incident light (obtainedvia filtering a broadband source or from a narrowband laser) isused to excite fluorophores and the emerging fluorescencespectrum at each excitation wavelength is detected