Principles of Spectrophotometry

Much of what we know about the photosynthetic apparatus was learned through spectroscopythat
is, measurements of the interaction of light and molecules. Spectrophotometry is an important branch
of spectroscopy that focuses on the technique of measurement. Here we will examine four topics:
Beer's law, the measurement of absorbance, action spectra, and difference spectra.

Beer's Law

An essential piece of information about any molecular species is how much of it is present.
Quantitative measures of concentration are one of the cornerstones of biological science. Of all the
methods that have been devised for measuring concentration, by far the most widely applied is
absorption spectrophotometry. In this technique, the amount of light that a sample absorbs at a
particular wavelength is measured and used to determine the concentration of the sample by
comparison with appropriate standards or reference data. The most useful measure of light absorption
is the absorbance (A), also commonly called the optical density (OD) (Web Figure 7.1.A). The
absorbance is defined asA = log I0 / I where I0 is the intensity of light that is incident on the sample
and I is the intensity of light that is transmitted by the sample.

Web Figure 7.1.A Definition of absorbance. A monochromatic incident light beam of

intensity I0 traverses a sample contained in a cuvette of length (l). Some of the light is absorbed by
the chromophores in the sample, and the intensity of light that emerges is I.

The absorbance of a sample can be related to the concentration of the absorbing species
through Beer's law:

A = cl

where c is concentration, usually measured in moles per liter; l is the length of the light path, usually
1 cm; and is a proportionality constant known as the molar extinction coefficient, with the units of
liters per mole per centimeter. The value of is a function of both the particular compound being
measured and the wavelength. Chlorophylls typically have an value of about 100,000 L mol 1 cm1.
When more than one component of a complex mixture absorbs at a given wavelength, the
absorbances due to the individual components are generally additive.
The Spectrophotometer

The absorbance is measured by an instrument called a spectrophotometer (Web Figure 7.1.B). The
essential parts of a spectrophotometer include a light source, a wavelength selection device such as a
monochromator or filter, a sample chamber, a light detector, and a readout device, usually also
include a computer, which is used for storage and analysis of the spectra. The most useful machines
scan the wavelength of the light that is incident on the sample and produce, as output, spectra of
absorbance versus wavelength, such as those shown in textbook Figure 7.7.

Web Figure 7.1.B Schematic diagram of a spectrophotometer. The instrument consists of a light
source, a monochromator that contains a wavelength selection device such as a prism, a sample
holder, a photodetector, and a recorder or computer. The output wavelength of the monochromator
can be changed by rotation of the prism; the graph of absorbance versus wavelength is called a
spectrum.

Action Spectra

The use of action spectra has been central to the development of our current understanding of
photosynthesis. An action spectrum is a graph of the magnitude of the biological effect observed as a
function of wavelength. Examples of effects measured by action spectra are oxygen evolution (Web
Figure 7.1.C) and hormonal growth responses due to the action of phytochrome (see Chapter 16 of
the textbook). Often an action spectrum can identify the chromophore responsible for a particular
light-induced phenomenon. Action spectra were instrumental in the discovery of the existence of the
two photosystems in O2-evolving photosynthetic organisms.
Web Figure 7.1.C An action spectrum compared to an absorption spectrum. The absorption
spectrum is measured as shown in Web Figure 7.1.B. An action spectrum is measured by plotting a
response to light such as oxygen evolution, as a function of wavelength. If the pigments used to
obtain the absorption spectrum are the same as those that cause the response, the absorption and
action spectra will match. In the example shown here, the action spectrum for oxygen evolution
matches the absorption spectrum of intact chloroplasts quite well, indicating that light absorption by
the chlorophylls mediates oxygen evolution. Discrepancies are found in the region of carotenoid
absorption, from 450 to 550 nm, indicating that energy transfer from carotenoids to chlorophylls is not
as effective as energy transfer between chlorophylls.

Some of the first action spectra were measured by T. W. Engelmann in the late 1800s (Web Figure
7.1.D). Engelmann used a prism to disperse sunlight into a rainbow that was allowed to fall on an
aquatic algal filament. A population of O2-seeking bacteria was introduced into the system. The
bacteria congregated in the regions of the filaments that evolved the most O2. These were the regions
illuminated by blue light and red light, which are strongly absorbed by chlorophyll. Today, action
spectra can be measured in room-sized spectrographs in which the scientist enters a huge
monochromator and places samples for irradiation in a large area of the room bathed by
monochromatic light. But the principle of the experiment is the same as that of Engelmann's
experiments.
Web Figure 7.1.D Schematic diagram of the action spectrum measurements by T. W. Engelmann.
Engelmann projected a spectrum of light onto the spiral chloroplast of the filamentous green
alga Spirogyra and observed that oxygen-seeking bacteria introduced into the system collected in the
region of the spectrum where chlorophyll pigments absorb. This action spectrum gave the first
indication of the effectiveness of light absorbed by accessory pigments in driving photosynthesis.

Difference Spectra

An important technique in studies of photosynthesis is light-induced difference spectroscopy, which

measures changes in absorbance (Web Figure 7.1.E). In this technique, bright light, often called
actinic light, is used to illuminate a sample, while a dim beam of light is used to measure the
absorbance of the sample at wavelengths other than that of the actinic beam. In this way a difference
spectrum is obtained, which represents the changes in the absorption spectrum of the sample induced
by illumination with the actinic light. Absorption bands that disappear upon illumination appear as
negative peaks; new bands that appear upon illumination appear as positive peaks. Difference spectra
give important clues to the identity of molecular species participating in the photoreactions of
photosynthesis. The difference spectrum of the photooxidation of P700 (a chlorophyll that absorbs
light of wavelength 700 nm, see textbook Figure 7.19) is shown in Web Figure 7.1.F (Ke 1973).
Web Figure 7.1.E Principle of difference spectroscopy. We measure difference spectra by observing
as a function of time the change in the absorbance of a measuring light 1 in a sample when an actinic
light is turned on. The actinic light 2 causes chemical changes in the sample that change its
absorption spectrum. Blocking filters are necessary to prevent scattered actinic light from entering the
detector. The up and down arrows signify the times when the actinic light was turned on and off,
respectively. The change in absorbance induced by the actinic light, A, can be either positive or
negative. Usually, measurements are made at one wavelength at a time. A difference spectrum is built
up by repetition of the measurement at many different wavelengths.

Web Figure 7.1.F Light-minus-dark difference spectrum for photooxidation of P700, measured as
shown in Web Figure 7.1.E. The decreases of absorption (bleaching) at 430 and 700 nm are due to
loss of the absorbance of P700. Increases observed around 450 nm and beyond 730 nm are due to
absorption by P700+ (oxidized P700). (After Ke 1973.)

By the use of special flash techniques, it is possible to record the difference spectrum at a given time
after flash excitation. Multiple difference spectra recorded at different times after flash excitation can
be used to measure the kinetics of the chemical reactions that follow photon excitation of a reaction
center. These techniques can have extraordinary time resolution, in some cases less than a
picosecond (1012 s), and have provided great insights into the earliest events in the photosynthetic
energy storage process.
Ilocano Folk Song

"Manang Biday"

Manang Biday, ilukatmo man

Ta bintana ikalumbabam
Ta kitaem toy kinayawan
Ay, matayakon no dinak kaasian

Siasinnoka nga aglabaslabas

Ditoy hardinko pagay-ayamak
Ammom ngarud a balasangak
Sabong ni lirio, di pay nagukrad

Denggem, ading, ta bilinenka

Ta inkanto diay sadi daya
Agalakantot bungat mangga
Ken lansones pay, adu a kita

No nababa, dimo gaw-aten

No nangato, dika sukdalen
No naregreg, dika piduten
Ngem labaslabasamto met laeng

Daytoy paniok no maregregko

Ti makapidut isublinanto
Ta nagmarka iti naganko
Nabordaan pay ti sinanpuso

Alaem dayta kutsilio

Ta abriem toy barukongko
Tapno maipapasmo ti guram
Kaniak ken sentimiento
1. Nagtago si Pedro, labas ang ulo. (Pedro hides but you can still see his head. )
2. Hindi pari, hindi hari, nagdadamit ng sari-sari. (Not a priest, not a king but wears different kinds of clothes.)
3. Bugtong-pala-bugtong, kadenang umuugong. (Riddle me, riddle me, here comes a roaring chain).
4. Heto na si Kaka, bubuka-bukaka. (Here comes Kaka, walking with an open leg.)
5. Buhok ni Adan, hindi mabilang. (Adam's hair, you can't count.)
6. Bibingka ng hari, hindi mo mahati. (Rice cake of the king, that you cannot divide.)
7. Sa araw ay bungbong, sa gabi ay dahon. (Roll in the morning, leaf in the afternoon).
8. Iisa ang pasukan, tatlo ang labasan. (It has one entrance, but has three exit. )
9. Malaking supot ni Mang Jacob, kung sisidlan ay pataob. (Big SquareBag of Mr Jacob, to use it, you have to turn it upside down)
10. Dalawang pipit nag titimbangan sa isang siit. (Two birds, trying to balance in one twig.)
11. Hayan na, hayan na di mo pa makita. (It's here, its here, but you can not see)
12. Baka ko sa Maynila, hanggang dito, dinig ang unga. (My cow in Manila, you can hear his moo).
13. Nagdaan si Kabo Negro, namatay na lahat ang tao. (General Negro pass by and eveybody die.)
14. Ako ay may kaibigan, kasama ko kahit saan. (I have a friend and he is with me everywhere I go).

15. Ang alaga kong hugis bilog, barya-barya ang laman-loob. ( I have a pet, his body is full of coins).
16. Sa liwanag ay hindi mo makita. Sa dilim ay maliwanag sila. (I can't see it in the light but I can see it in the dark.)
17. Palda ni Santa Maria. Ang kulay ay iba-iba. (Maria's skirt, in different colours.)
18. Kaisa-isang plato, kita sa buong Mundo. ( One plate, can be seen around the world).
19. Nagsaing si Hudas, kinuha ang tubig itinapon ang bigas. (Judas cooked the rice, he took the water and throw the rice.)
20. Bahay ni Tinyente nag-iisa ang poste. (House of the Lieutenant,with only one post.)
21. May isang prinsesa, nakaupo sa tasa. (A princess sitting in a cup)

22. Ate mo, ate ko, Ate ng lahat ng tao. (My sister, your sister, everyone's sister)
23. Hiyas na puso, kulay ginto, mabango kung amuyin, masarap kung kainin. (Shape like a heart, gold in color, sweet to smell and good
to eat.)
24. Butong binalot ng bakal, bakal na binalot ng kristal. (Seed that is wrap in steel, steel that is wrap in crystal).
25. Nag tapis nang nag tapis nakalitaw ang bulbolis. (She wears a skirt, but you can still what is inside).
26. Aling pagkain sa mundo, ang nakalabas ang buto? (What fruit in the world that the seed is out?)
27. Heto na si Ingkong, nakaupo sa lusong. (Here comes Ingkong, sitting in a fish catcher.)
28. Nakatalikod na ang prinsesa, mukha niya'y nakaharap pa. (The princess is on her back, but her head is still facing us)
29. Balat niya'y berde, buto niya'y itim,laman niya'y pula, sino siya? (Her skin is green, her seed is black, her tissue is red, who is she?)
30. Kung tawagin nila'y santo, hindi naman milagroso. (He is called Saint, but with no miracle.)
31. Bahay ni Mang Pedro, punung-puno ng bato. (House of Pedro, full of stone)
32. Baboy sa pulo, ang balahibo ay pako. (An island pig with a hair as hard as a nail.)

33. Nanganak ang birhen, itinapon ang lampin. (The virgin gave birth, but throw the nappy)
34. Nakayuko ang reyna di nalalaglag ang korona. (The queen tilt her head but the crown did not fall)
35. May langit, may lupa, May tubig, walang isda. (There is a sky, there is soil, there is water, but no fish)
36. Kumpul-kumpol na uling, hayon at bibitin-bitin. (A bunch of charcoal, hanging here and there.)
37. Bunga na ay namumunga pa. (A fruit that still bears fruit)
38. Tiningnan nang tiningnan. Bago ito nginitian. (It was look twice before it smile)
39. Hindi prinsesa, hindi reyna. Bakit may korona? (Not a princess, not a queen, but wears a crown).
40. Isang magandang dalaga.Di mabilang ang mata. (A beautiful girl, you can't count her eyes
1. Pako - (Nails)
2. Sampayan - (Clothesline)
3. Tren - (Train)
4.Gunting - (Scissors)
5. Ulan - (Rain)
6. Tubig - (Water)
7. Banig - (Mat)
8. Damit/Baro - (Dress)
9. Kulambo - (Mosquito Net)
10. Hikaw - (Earrings)
11. Hangin - (Wind)
12.Kulog - (Thunder)
13. Gabi - (Night)
14. Anino - (Shadow)
15. Alkansiya - (Money Box)
16. Bituin - (Star)
17. Bahaghari - (Rainbow)
18. Buwan - (Moon)
19. Gata ng Niyog - (Coconut Milk)
20. Payong - (Umbrella)
21. Kasoy (Cashew)
22. Atis (Sugar Apple)
23. Mangga (Mango)
24. Lansones (Lanzones)
25. Mais (Corn)
26. Kasoy (Cashew)
27. Kasoy (Cashew)
28. Balimbing (Star Apple)
29.Pakwan (Watermelon)
30. Santol (Santol fruit)
31. Papaya (Pawpaw)
(House of Pedro, full of ston
32. Langka (Jackfruit)
(An island pig with a hair as hard as a nail.)
33. Saging (Banana)
34. Bayabas (Guava)
35. Niyog (Coconut)
36. Duhat (Black Plum)
37. Bunga
38. Mais (Corn)
39. Bayabas (Guava)
40. Pinya (Pineapple)
(A beautiful girl, you can't count her eyes)
Riddles in Tagalog is referred to as Bugtong. Ito 13. Baka ko sa palupandan, ungay nakakarating
ay isang palaisipan o talinghaga na may kahit saan.
nakatagong kahulugan. Ang bugtong ay Isang Sagot: kulog
pangungusap na patanong nahumihingi ng 14. May bintana ngunit walang bubungan,
kasagutan. Ang mga bugtong ay kadalasang may pinto ngunit walang hagdanan.
patungkol sa pag-uugali, kaisipan, pang-araw-araw Sagot: kumpisalan
na buhay at katutubong paligid ng mga Pilipino. 15. Heto na si Kaka, bubuka-bukaka.
Mga halimbawa ng Bugtong na may Sagot: Sagot: palaka
1. Maikling landasin, di maubos lakarin. 16. Magandang prinsesa, nakaupo sa tasa.
ANINO (SHADOW) Sagot: kasoy
2. Hindi hayop, hindi tao, pumupulupot sa tiyan 17. Hindi pari, hindi hari, nagdadamit ng sari-
mo. sinturon (belt) sari.
3. Dala mo dala ka, dala ka ng iyong dala. Sagot: paruparo
sapatos (shoes) 18. Dalawang batong itim, malayo ang nararating.
4. Maliit pa si Kumpare, nakakaakyat na sa tore. Sagot: mga mata
langgam (ant) 19. Kay lapit-lapit na sa mata, di mo pa rin
5. Kung gusto mong tumagal pa ang aking buhay, makita.
kailangang ako ay mamatay. kandila (candle) Sagot: tenga
. 20. Sa maling kalabit, may buhay na kapalit.
Karagdagang Mga Bugtong Sagot: baril
1. Kung kailan mo pinatay, saka pa humaba ang 21. Sa buhatan ay may silbi, sa igiban ay walang
buhay. sinabi.
Sagot: kandila Sagot: bayong o basket
2. Baboy ko sa pulo, ang balahiboy pako. 22. Hindi tao, hindi hayop, kung uminom ay
Sagot: langka salup-salop.
3. Nang sumipot sa maliwanag, kulubot na ang Sagot: batya
balat. 23. Isa ang pasukan, tatlo ang labasan.
Sagot: ampalaya Sagot: kamiseta
4. Isang butil ng palay, sakot ang buong buhay. 24. Butot balat na malapad, kay galing kung
Sagot: ilaw lumipad.
5. Ako ay may kaibigan, kasama ko kahit saan. Sagot: saraggola
Sagot: anino 25. Lumuluha walang mata, lumalakad walang
6. Sa araw ay bungbong, sa gabi ay dahon. paa.
Sagot: banig Sagot: ballpen o Pluma
7. Dumaan ang hari, nagkagatan ang mga pari. 26. Nagbibigay na, sinasakal pa.
Sagot: siper Sagot: bote
8. Munting hayop na pangahas, aaligid-aligid sa 27. May puno walang bunga, may dahon walang
ningas. sanga.
Sagot: gamu-gamo Sagot: sandok
9. Tinaga ko ang puno, sa dulo nagdurugo. 28. Hinila ko ang baging, sumigaw ang matsing.
Sagot: gumamela Sagot: kampana o batingaw
10. Naabot na ng kamay, ipinagawa pa sa tulay. 29. Yumuko man ang reyna, di malalaglag ang
Sagot: kubyertos korona.
11. Malaking supot ni Mang Jacob, kung sisidlan Sagot: bayabas
ay pataob. 30. Nakatalikod na ang prinsesa, ang mukhay
Sagot: kulambo nakaharap pa.
12. Maliit pa si kumare, marunong ng humuni. Sagot: balimbing
Sagot: kuliglig