 The basis of most spectrophotometry is the concept in figure 1: when one inputs energy into a molecular bond, it becomes excited.

To make this easier to visualize; we can assume molecular bond as a spring. The two balls connected by the spring are molecules, such as hydrogen, or any kind of molecule really; for ease of explanation, let’s call these balls and spring a system.  Generally speaking, since room temperature (or any temperature above 0 K, or absolute zero) will always give the system energy bigger than 0 (through heat; since heat is basically a kinetic energy on the molecular level, we can talk about this more if you want.), the balls and spring will always contract and expand. If energy is put into the system, it’s as if we’re pulling the two balls apart even more, therefore increasing the frequency of contraction and expansion; just like every spring we can see in life. How do we put energy into the system? In the case of Spectrophotometry, it’s most likely some kind of light, whether it’s UV + Visible light (UV-Vis Spec), Infra red (IR-Spec), or (if I’m not mistaken) a wide range, or continuum of wavelength (Atomic Absorption Spec). To make things a little easier, in figure 2 the increase in energy is represented in energy level diagram; The Y-axis represents Energy level.  To go from E1 à E2, one has to put in energy. The difference between E1 and E2 is ΔE. In the case for all spectrophotometry, ΔE is always bigger than 0; that is to say that energy will always shift upwards, or in other words, excitation always happen. Since the energy source in spectophotometry  is almost always light, we can represent  ΔE as: ΔE = hv. ΔE = hv is the equation that relates energy to the frequency of the light; h in this case is a constant called the plank’s constant, while v is the frequency of light. But we never express light in terms of frequency you said? Correct!. We express light in terms of wavelength (nanometers; nm). To relate frequency to wavelength there’s a simple equation: v = c/λ, where c is the speed of light (3x108m/s), and λ is the wavelength. All of these equations are hard to memorize you say? Again, correct. Though E = hv is a very commonly used equation, remembering v = c/ λ can be hard. The trick is, since the unit for v (frequency) is 1/sec, and c is m/sec, and wavelength is m (or nm), in order to get 1/sec, c (m/sec) has to be the nominator (the one on the top), while wavelength (m) has to be the denominator (the one on the bottom). I hope that helps a little.  Another idea is the idea that different molecular bonds require very different specific wavelength to excite. Why? Think back to the energy level diagram; different structure excitation have a very specific energy level gap, that is the energy required to excite a specific structure is, again, very specific. And if we need a specific energy, according to E = hv, we need a specific frequency. If we need a specific frequency, according to v = c/ λ, we require a very specific wavelength; this is why different molecules require different wavelength to excite, such as 260 nm to excite DNA.

Now enough of this mumbo jumbo about excitation and the planck dude, let’s move on to actual spectrophotometry. 