Column Chromatography is another common and useful separation technique in organic
chemistry. This separation method involves the same principles as TLC, but can be applied to separate larger quantities than TLC. Column chromatography can be used on both a large and small scale. The applications of this technique are wide reaching and cross many disciplines including biology, biochemistry, microbiology and medicine. Many common antibiotics are purified by column chromatography.
To understand to uses of this separation technique, we can use the last experiment as an
example. In the TLC experiment, we separated and analyzed the different components that makeup over-the-counter painkillers. The technique of TLC was useful in determining the type and number of ingredients in the mixture, but it was not helpful for collecting the separated components. We could only separate and visualize the spots. If we needed to collect the separated materials, column chromatography could be used. We could load 100 mg of a crushed Anacin tablet on a column made up of a silica stationary phase and separate the aspirin from the caffeine and collect each of these compounds in separate beakers. Column chromatography allows us to separate and collect the compounds individually. In this experiment, Column Chromatography
(abbreviated CC) will be used to separate the starting material from the product in the oxidation of fluorene to flourenone and TLC will be used to monitor the effectiveness of this separation.

Choosing a Stationary Phase
As with TLC, alumina and silica are the two most popular stationary phases in column
chromatography. For these common phases, the partitioning works in an analogous manner. The more polar sample will be retained on the stationary phase longer. Thus the least polar compound will elute from the column first, followed by each compound in order of increasing polarity.

Although the interactions between the mobile and stationary phase are based on the same
principles for CC and TLC, be careful when predicting the order of elution. Since the direction of the solvent flow in TLC moves up and in CC the solvent flows down, it appears that the order is “upside-down”. In TLC the more polar molecules will have lower Rf values, but in CC they will be retained longer on the column.Remember this when considering the polarities of the stationary phase as well as the polarity of the compounds being separated when predicting the order of elution.
Stationary phases for CC can come in a variety of sizes, activities, acidic and basic variations for both alumina and silica. The types of stationary phase chosen are determined experimentally, or often based on results from a previous TLC experiment. The type of adsorbent, the size of the column, the polarity of the mobile phase as well as the rate of elution all affect the separation. These conditions can be manipulated to get the best separation for your mixture.

Choosing Solvents
Solvent systems for use as mobile phases in CC can be determined from previous TLC
experiments, the literature, or experimentally. Normally, a separation will begin by using
nonpolar or low polarity solvent, allowing the compounds to adsorb to the stationary phase, then SLOWLY switching the polarity of the solvent to desorb the compounds and allow them to travel with the mobile phase. The polarity of the solvents should be changed gradually. On a macroscale, the mixing of two solvents can create heat and crack the column leading to a poor separation.
Some typical solvent combinations are ligroin-dichloromethane, hexane-ethyl acetate and hexane-toluene. Often an experimentally determined ratio of these solvents can sufficiently separate most compounds. Solvents such as methanol and water are normally not used because they can destroy the integrity of the stationary phase by dissolving some of the silica gel.

Columns can be as thin as a pencil to a diameter of several feet in industrial processes. They can separate milligram to kilogram quantities of materials. In this experiment, we will be separating a mixture of approximately 50 mg, so a small column can be used. Figure 8.1 shows the typical set-up we will be using during this experiment. It is essential to have several clean tared Erlenmeyer flasks, reaction tubes, beakers, test tubes or vials available to collect the solvent and compounds as they elute. Once you have the general set-up prepared, you can move on to packing the stationary phase in the column.

Packing the Column
There are several acceptable methods when packing a column. These include dry packing
(there are two versions of dry packing discussed here) and the slurry method. The slurry method normally achieves the best packing results, but there are several occasions when the dry packing method works just as well if not better.
Dry packing is the method of choice for a microscale column. Begin by filling the column with a nonpolar solvent. Slowly add the powdered alumina or silica while gently tapping the side of the column with a pencil. The solid should “float” to the bottom of the column. Try to pack the column as evenly as possible; cracks, air bubbles, and channels will lead to a poor separation.

For the second dry pack method, the stationary phase is deposited in the column before the solvent. In this case fill the column to the intended height with the stationary phase and then slowly add the nonpolar solvent. The solvent should be added slowly as to avoid uneven channeling. This method is typically used with alumina only, since silica gel expands and does not pack well with this dry method.
The slurry method is often used for macroscale separations. Combine the solid stationary
phase with a small amount of nonpolar solvent in a beaker. Thoroughly mix the two until a
consistent paste is formed, but is still capable of flowing. Pour this homogeneous mixture into the column as carefully as possible using a spatula to scrape out the solid as you pour the liquid. The slurry method normally gives the best column packing, but is also a more difficult technique to master. Whether the dry or slurry method is chosen, the most important aspect of packing the column is creating an evenly distributed and packed stationary phase. As mentioned, cracks, air bubbles and channeling will lead to a poor separation.
Once the column is loaded, open the stopcock and allow the solvent level to drop to the top of the packing, but do not allow the solvent layer to go below this point. Allowing this solvent level to go below the stationary phase, (known as letting the column to “run dry,”) should always be avoided. Since it allows air bubbles and channel formation to occur leading to a poor separation.

Adding the Sample
Once the packing is complete, the sample can be loaded directly to the top of the column.
Normally, a minimum amount of a polar solvent, 5-10 drops, is used to dissolve the mixture. The solution is then carefully added to the top of the column using a pipet without disrupting the flat top surface of the column. A thin horizontal band of sample is best for an optimal separation. After the sample is loaded, a small layer of white sand is added to the top of the column. This will help to keep the top of the column level when adding solvent eluent. Once the mixture is added and the protective layer of sand is in place, continuously add the solvent eluent while collecting small fractions at the bottom of the column. Using a pipet to add the first bit of solvent on top of the packing, sample, and sand will minimize disturbance of the column and diluting the sample. Collecting small fractions (1-3 mL) is important to the success of your column separation.

Fractions that are too small can always be pooled together; however, if the collected fractions are too large, you may get more than one compound in any particular fraction. If this occurs, the only way to complete the separation is to redo the chromatography. Since column chromatography is time consuming, collecting large fractions is discouraged.

Monitoring the Column
If the mixture to be separated contains colored compounds, then monitoring the column is very simple. The colored bands will move down the column along with the solvent and as they approach the end of the column, collect the colors in individual containers. Use the color as your guide. However, most organic molecules are colorless. In this case, the reaction must be monitored by TLC. Spot each fraction on a TLC plate. Four or five fractions can be spotted on a single TLC plate. Develop the plate and use the observed spot or spots to determine which compound is in each of the collected fractions. Spotting some of the starting material or the product (if available) on the TLC plate as a standard will help in the identification.

Isolating the Separated Compounds
Once you believe all the materials have been removed from the column, the colors of the
materials or TLC results should indicate which fractions contain the compound(s) you are
interested in isolating. Combine the like or same fractions and evaporate the solvent. The pure separated compound will be left behind. Recrystallization may be used to further purify a solid product. However, on a milligram scale, there is usually not enough material to do this.

Flash Chromatography
Column chromatography is often very time consuming. Allowing the solvent to elute through the column one drop at a time takes patience. One method to speed up the process is to use Flash Chromatography. This method uses a pressure of about 10 psi of air or nitrogen to force the mobile phase through the column. Because the rate of the mobile phase is increased, in general, this method gives a poorer separation. However, by using a finer grade of alumina or silica, flash chromatography can be used to increase the speed without lowering the quality of the separation.

Reverse-Phase Chromatography
HPLC (High Performance Liquid Chromatography) is a variation on the traditional liquid
chromatographic methods. High pressure pumps are used to force solvent through a tightly packed column connected to a variety of different types of very sensitive detectors. Modern HPLC is used extensively in biochemistry to separate cellular components such as proteins, lipids, and nucleic acids. Mixtures of these types require aqueous mobile phases such as methanol-water or acetonitrile-water and these liquids do not work well on normal silica or alumina stationary phases. Instead of these polar phases, very nonpolar ones, called “reverse-phase” packing are used. These are manufactured by bonding lots of hydrocarbon molecules to the surfaces of a silica gel particles so that the silica gel is like a very nonpolar “grease ball.” In this situation, the order of elution will be exactly opposite the behavior on an alumina or silica column. On a reverse-phase column, the more nonpolar materials will adhere to the stationary phase (or like material) longer
and the polar compounds will elute first.

Chiral Separations
Many compounds can be separated using typical chromatographic stationary phases and
solvents. These separations depend on the difference in polarity of the molecules to be separated. However, how would you separate two compounds with the identical polarity, such as enantiomers? This separation technique is of great importance in the pharmaceutical industry where the FDA controls the amounts of impurities, including enantiomers, in prescribed drugs. For example, Thalidomide, a drug administered in the 60’s has two enantiomers. This drug was used as a sedative and an anti-depressant, but was found to cause abnormalities in the fetuses of pregnant
women. Although this drug was pulled from the market due to the resulting birth defects, there is recent literature that suggests that only one of the enantiomers caused the defects. If the enantiomers could be completely separated, Thalidomide might be used as an FDA approved drug and be helpful to people today.
Chiral stationary phases can be used to separate enantiomers. By giving the stationary phase a “handedness,” one enantiomer will be specifically retained on the column. These columns are very expensive and specific to the particular type of separation, but have led to great achievements in separation science.
There are many, many different types of chromatographic methods including gel electrophoresis and size exclusion chromatography that have not been discussed here. Hopefully, with the basic chromatographic background provided, you can apply this knowledge to the many different types of chromatography used in many different professions today.



Column Chromatography

  1. Objective

The aim of this experiment is to separate two substances using column chromatography. As an example, methylene blue and methyl orange will be separated using an alumina packed column. The separated substances will then be analyzed spectrophotometrically using a visible spectrophotometer.

  1. Principle and Theory

Chromatography is a technique in which compounds to be separated are distributed between a mobile phase and a stationary phase. In such a system, different distributions based on selective adsorption give rise to separation. There are different types of chromatography, such as paper, thin layer, or column chromatography, each with its own strengths and weaknesses.

Column chromatography is one of the most useful methods for the separation and purification of both solids and liquids when carrying out small-scale experiments. The separation can be liquid/solid (adsorption) or liquid/liquid (partition) in column chromatography. The stationary phase, a solid adsorbent, is usually placed in a vertical glass column and the mobile  phase, is added from the top and let flow down through the column by either gravity or external pressure (Figure 1).


Figure 1. Column chromatography involves a mobile phase flowing over a stationary phase.

Column chromatography is advantageous over most other chromatographic techniques because it can be used in both analytical and preparative applications. It can be used to determine the number of components of a mixture and as well as the separation and purification of those components.

Column chromatography isolates desired compounds from a mixture in such a way that the mixture is applied from the top of the column.  The columns are usually glass or plastic with sinter frits to hold the packing. The liquid solvent (eluent) is passed through the column by gravity or by the application of air pressure. The eluent, instead of rising by capillary action up a thin layer, flows down through the column filled with the adsorbent. Equilibrium is established between the solute adsorbed on the adsorbent and the eluting solvent flowing down through the column. Stationary phases are almost always adsorbents. Adsorbent is a substance that causes passing molecules or ions to adhere to the surface of its particles. The mobile phase is a solvent that flows past the stationary phase, dissolving the molecules of the compounds to be separated some of the time.

Because the different components in the mixture have different interactions with the stationary and mobile phases, they will be carried along with the mobile phase to varying degrees and a separation will be achieved. The individual components, or elutants, are collected as solvent drips from the bottom of the column.

Many compounds are not visible to the eye when dissolved in a solvent or adsorbed on a adsorbent. Visualization processes make these substances visible. The used techniques for this purpose include UV lights that cause fluorescence or phosphorescence and chemical reactions that give colored compounds.

2.1. Adsorbent

Silica gel (SiO2) and alumina (Al2O3) are two adsorbents commonly used by organic chemists for column chromatography. These adsorbents are sold in different mesh sizes, indicated by a number on the bottle label: “silica gel 60” or “silica gel 230-400” are a couple of examples. This number refers to the mesh of the sieve used to size the silica, specifically, the number of holes in the mesh or sieve through which the crude silica particle mixture is passed in the manufacturing process. If there are more holes per unit area, those holes are smaller, thus only smaller silica particles are allowed to pass the sieve. The larger the mesh size, the smaller the adsorbent particles are.

Adsorbent particle size affects the way the solvent flows through the column. Smaller particles (higher mesh values) are used for flash chromatography; larger particles (lower mesh values) are used for gravity chromatography.

Alumina is quite sensitive to the amount of water which is bound to it; the higher its water content, the less polar sites it has to bind organic compounds, and thus the less “sticky” it is. This stickiness or activity is designated as I, II, or III with I being the most active. Alumina comes in three forms: acidic, neutral, and basic. The neutral form of activity II or III, 150 mesh, is most commonly employed.

2.2. Solvent

The polarity of the solvent, which is passed through the column, affects the relative rates at which compounds move through the column. Polar solvents can more effectively compete with the polar molecules of a mixture for the polar sites on the adsorbent surface and will also better solve the polar constituents. Consequently, a highly polar solvent will move even the highly polar molecules rapidly through the column. If a solvent is too polar, movement becomes too rapid, and little or no separation of the components of a mixture will result. On the other hand, if a solvent is not polar enough, no compounds will elute from the column. Proper choice of an eluting solvent is thus crucial for a successful application of column chromatography as a separation technique since compounds interact with the silica or alumina largely due to polar interactions.

2.3. Elution Chromatography

Elution involves transporting a species through a column by continuous addition of fresh mobile phase. Single portion of sample contained in the mobile phase is introduced from the top of the column whereupon the components of that sample distribute themselves between two phases. Introduction of additional mobile phase (the eluent) forces the solvent containing a part of the sample down the column where further partition between the mobile phase and fresh portions of the stationary phase occurs (time t1). Simultaneously, partitioning between the fresh solvent and the stationary phase takes place at the site of the original sample. Continued additions of solvent carry solute molecules move down the column in a continuous series of transfers between the mobile and the stationary phases. Because the movement of the sample can occur in the mobile phase, however, the average rate at which a solute zone migrates down the column depends upon the fraction of time it spends in that phase. This fraction is small for solutes that are strongly retained by the stationary phase and it is large where retention in the mobile phase is more likely. Ideally the resulting differences in rates cause the components in the mixture to separate into bands, or zones located along the length of the column (t2). Isolation of the separated species is then accomplished by passing a sufficient quantity of mobile phase through the column to cause the individual zones to pass out the end, where they can be detected or collected ( times t3 and t4) (Figure 2).

2.3.1. Chromatograms

If a detector that responds to the presence of analyte (dye) is placed at the end of the column and its signal is plotted as a function of time (volume of added mobile phase), a series of peaks is obtained. Such a plot, called a chromatogram, is useful for both qualitative and quantitative analysis.

2.3.2. Migration Rates of Solutes The Partition Coefficient

An analyte is in equilibrium between the two phases;


where the equilibrium constant K is called the partition coefficient.

CS: molar concentration of analyte in stationary phase

CM: molar concentration of analyte in mobile phase


figure 2. Diagram showing the separation of a mixture of components A and B by column chromatography Retention Time

The time it takes after sample injection for the analyte peak to reach the detector is called



Paul C. 2002. “The Essence of Chromatography,” Elsevier. (e-book,

Skoog D. A. and Leary J. J. 1991. “ Principles of Instrumental Analysis”, 4th Edition, Saunders Collage Publishing.

Preparing your own thin layer chromatography plates (and then using them)


Image Notes

1.These were made with lab grade silica gel, on glass slides, with plaster of paris as the binder

2.  These were made with silica gel from dessicator packets, prepared in the same way as above but with less suspension to work with (hence the gaps near the edge)

step 1: Gather the materials

First off you need some basic supplies:

  1. an oven, generally comes with houses
  2. a weigh scale, nothing too fancy should be accurate to one tenth of a gram, e.g This digital scale from Amazon an old plastic bottle you don’t care about, not too large (I used a 150mL one)
  3. a pan, for resting the plates on and for putting in the oven.
  4. a mortar and pestle, larger ones are easier to work with than smaller ones
  5. a syringe, 10cc minimum, plastic works fine, I got mine from Home Depot
  6. glass slides, you can also use sheets of tin or plastic, basically anything stiff that won’t interact with water

Then you need your “chemicals” for preparing the slides:

  1.  Anhydrous Calcium Sulfate, a.k.a. Plaster of Paris, I liberated mine from an artsy friend
  2. Water, from the tap, or distilled if impurities are an issue
  3. Silica Gel – This is the desiccant in those little packets you find in medicine bottles and assorted what-nots.

 Note: Silica Gel is hygroscopic, and its fine particles can be harmful if inhaled. It is not a bad idea to wear gloves and a mask while grinding this stuff.

step 2: Weigh out and mix the silica gel and plaster of paris

The plaster of paris is the binder and is present only to keep the silica from sloughing off the glass slides. I have found 10-20% (by weight) plaster of paris in the final mix works well.

For this project, weigh out:

  •  1.0g plaster of paris
  • 4.0g silica gel (ground)

Combine these in the mortar and pestle and grind together very well. The mixture should be very homogeneous and the finer the particles the better the separation.

step 3: Suspend the powder in water

Transfer the powder into an old plastic bottle that you will never want to use for anything else again (especially if you let the plaster set before cleaning it out and are as lazy as I).

Add 10mL water (or a water to powder ratio of 2:1), the syringe is handy for this.

Cap the bottle and shake violently for one minute, the bottle that is. The goal is to form a slurry of all the solid in water


step 4: Coat glass slides with suspension

Draw up the newly formed suspension into the syringe.

With the slides cleaned and dried (and free of fingerprints or oils), move the tip of the syringe back and forth width-wise across the slide while applying gentle pressure to the plunger. The motion is sort of like tiling a field. You don’t want to pour or dispense it all at once as the layer will form a hill instead, and will be too thick. By going back and forth slowly you can dispense a reasonably even, thin, layer of suspension across the plate.

The thickness of the layer is important, less than 1mm when dry is preferable so be careful not to overdo it.



step 5: Air dry followed by activation

You want to air dry the slides to allow the plaster to set. Just leave the slides in a calm place for about an hour, or until they are white and smooth.

Prior to using, the tlc plates need to be activated. This entails driving off any remaining water that would still be held by the silica gel. This, apparently, frees-up the -OH groups of the silica gel to do your bidding as a stationary phase does.

Activation is done by heating the plates in an oven at 120C for 30-45 minutes. At this stage there really is no harmful chemical residues to worry about and this can be done in a regular household oven (unless the oven caretaker objects of course).

 After they have cooled the plates are ready for use so that you may elute to your heart’s content.

 The advantage of using glass slides is that when you’re done with the plate you can always scrape off the stationary phase, clean it, and re-layer it.

step 6: Some final notes on preparing the plates

These plates can be used like any other, though generally home-made plates have a more brittle stationary phase so be gentle. Tweezers are a good investment, and wield the “science tongs” carefully for with great power comes great responsibility.

I’ve heard that some silica gel desiccant packets contain fungicides and other chemical dopants, they may interfere with the operation of your plates. I have absolutely no advice on how to deal with this besides, perhaps, cleaning the silica powder before hand with a non-polar solvent. I did not have this problem and the plates I made from the desiccant packets worked the same as ones I made from lab grade silica gel (for chromatography, oooh).

Other stationary phases can be used as well, alumina for example. In this case slightly less water can be used, about 1.5:1 ratio instead of 2:1 as alumina does not absorb as much water as silica (and you want to maintain the consistency of the suspension). Cellulose can also be used, though I haven’t experimented with it, I hear that you don’t need to use a binder as cellulose is sticky enough on its own. You can, of course, experiment with your own stationary phases. There is a lot of literature out there, ripe for the googling.


you can also download homemade-tlc-plates



1 HCl 0,1 N Ukur ± 8,5 cc HCl pekat, encerkan sampai 1 liter
2 NaOH 0,1 N Timbang ± 40 gr NaOH larutkan dalam sampai 1 liter air
3 KOH 0,1 N Timbang 5,6 gr KOH larutkan sampai 1 liter air
4 KMnO4 0,1 N Timbang 3,2 gr kristal KMnO4 larutkan dalam air panas 100 cc, kemudian encerkan dengan air hangat sampai 1 liter, diamkan selama 24 jam (tidak boleh lebih dari 1 minggu)
5 Na2S2O3 0,1 N Timbang ± 25 gr garam Na2S2O3.5H2O, larutkan dalam 1 liter air dan tambahkan ± 0,1 gr Na2CO3 untuk stabilisator
6 I2 0,1 N Timbang 12,7 gr I2 dan larutkan 1 liter air dengan ± 40 gr KI
7 AgNO3 0,1 N Timbang dengan teliti 17 gr AgNO3 kristal dan larutkan dalam 1 liter air
8 KCNS 0,1 N Timbang ± 9,7 gr KCNS dan larutkan dalam 1 liter air
9 NH4CNS 0,1 N Timbang 3,7 gr NH4CNS, larutkan dalam 1 liter air
10 EDTA 0,1 N Timbang 37,23 gr EDTA dan larutkan dalam 1 liter air
11 ZnSO4 0,1 N Timbang 8,05 gr ZnSO4 dan larutkan sampai volume 500 cc
12 Karbonat loog Timbang 4 gr NaOH dan 5,3 gr Na2CO3 larutkan dalam 500 cc air
13 NaOH alkoholis Timbang 4 gr NaOH dan larutkan dalam 500 cc air dan 500 cc alcohol 96%
14 Buffer pH 10 142 ml larutan NH4OH pekat + 17,5 gr NH4Cl, encerkan sampai volume 250 cc


                      INDIKATOR ASAM BASA


1 PP Timbang 0,5 gr PP, larutkan dalam 60 cc alcohol 96% dan 40 cc air
2 MO Timbang 0,1 gr MO dan larutkan dalam 60 cc alcohol 96% dan 40 cc air
3 MR Timbang 0,1 gr MR dan larutkan dalam 60 cc alcohol 96% dan 40 cc air
4 K2CrO4 5% Timbang 5 gr K2CrO4 larutkan dalam 95 cc air
5 Garam Fe Timbang 2 gr Fero Amonium Sulfat NH4Fe(SO4)2.2H2O, larutkan dalam 100 cc air + HNO3 pekat
6 EBT Larutkan o,2 gr EBT dalam 15 ml etanol amin dan 5 ml etanol
7 amylum Timbang 5 gr amylum, tambahkan sedikit air dingin dan larutkan dengan 100 cc air mendidih dan tambahkan beberapa mgr HgI2
8 murexid Timbang 0,5 gr murexide, dibuat suspense dengan 100 cc air diambil bagian atasnya
9 Dimetil glioksin 1 gr dimetil glioksin dilarutkan dalam 99 gr alcohol ( bj alcohol 0,8 )
10 Larutan Wiys 15 gr Yod dilarutkan dalam 1 L larutan asam asetat 99% dan alirkan gas Cl2 diperlukan ± 3,6 gr Cl2, simpan dalam botol gelap dan disimpan tidak boleh lebih dari 1 bulan

Spektrofotometri UV-Vis

A. Pengertian Dasar Spektrofotometri UV-Vis

            Spektrofotometri merupakan salah satu metode dalam kimia analisis yang digunakan untuk menentukan komposisi suatu sampel baik secara kuantitatif maupun kualitatif yang didasarkan pada interaksi antara materi dengan cahaya. Peralatan yang digunakan dalam Spectrofotometri disebut spektrofotometer. Cahaya yang dimaksud dapat berupa cahaya visible, UV dan inframerah, sedangkan materi dapat berupa atom dan molekul namun yang lebih berperan adalah elektron valensi.

Sinar atau cahaya yang berasal dari sumber tertentu disebut juga sebagai radiasi elektromagnetik. Radiasi elektromagnetik yang dijumpai dalam kehidupan sehari-hari adalah cahaya matahari.

Dalam interaksi materi dengan cahaya atau radiasi elektromagnetik, radiasi elektromagnetik kemungkinan dihamburkan, diabsorbsi atau diemisikan sehingga dikenal adanya spektroskopi hamburan, spektroskopi absorbsi ataupun spektroskopi emisi.

Pengertian spektroskopi dan Spectrofotometri pada dasarnya sama yaitu di dasarkan pada interaksi antara materi dengan radiasi elektromagnetik. Namun pengertian Spectrofotometri lebih spesifik atau pengertiannya lebih sempit karena ditunjukan pada interaksi antara materi dengan cahaya (baik yang dilihat maupun tidak terlihat). Sedangkan pengertian spektroskopi lebih luas misalnya cahaya maupun medan magnet termasuk gelombang elektromagnetik.

Radiasi elektromagnetik memiliki sifat ganda yang disebut sebagai sifat dualistik cahaya yaitu:

  1. Sebagai gelombang
  2. Sebagai partikel-partikel energi yang disebut foton

Karena sifat tersebut maka beberapa parameter perlu diketahui misalnya panjang gelombang, frekuensi dan energi tiap foton. Panjang gelombang (λ) didefinisikan sebagai jarak antara dua puncak.

1Gambar 1 Panjang gelombang

(Sumber: Wiryawan, 2008)

Hubungan dari ketiga parameter di atas dirumuskan oleh Planck yang dikenal dengan persamaan Planck. Hubungan antara panjang gelombang dengan frekuensi dirumuskan sebagai

c = λ . vatau λ = atau v =

Persamaan Planck: hubungan antara energi tiap foton dengan frekuensi

E = h . v

E =


E = energi tiap foton

h = tetapan Planck (6,626 x 10-34 J.s)

v = frekuensi sinar

c = kecepatan cahaya (3 x 108 m.s-1)

Dari rumus di atas dapat diketahui bahwa energi dan frekuensi suatu foton akan berbanding terbalik dengan panjang gelombang tetapi energi yang dimiliki suatu foton akan berbanding lurus dengan frekuensinya. Misalnya: energi yang dihasilkan cahaya UV lebih besar dari pada energi yang dihasilkan sinar tampak. Hal ini disebabkan UV memiliki panjang gelombang (λ) yang lebih pendek (100–400 nm) dibanding panjang gelombang yang dimiliki sinar tampak (400–800 nm).

Interaksi antara materi dengan cahaya disini adalah terjadi penyerapan cahaya, baik cahaya UV, Vis maupun IR oleh  materi  sehingga  Spectrofotometri  disebut  juga sebagai spektroskopi absorpsi.

Dari empat jenis Spectrofotometri ini (UV, Vis, UV-Vis dan IR) memiliki prinsip kerja yang sama yaitu adanya interaksi antara materi dengan cahaya yang memiliki panjang gelombang tertentu. Perbedaannya terletak pada panjang gelombang yang digunakan.

Secara sederhana Instrumen Spectrofotometri yang disebut spektrofotometer terdiri dari:

sumber cahaya – monokromator – sel sampel – detektor – read out (pembaca)

2Gambar 2 Instrumen Spectrofotometri

(sumber: Wiryawan, 2008)

Fungsi masing-masing bagian:

  1. Sumber sinar polikromatis berfungsi sebagai sumber sinar polikromatis dengan berbagai macam rentang panjang gelombang. Untuk sepktrofotometer.
  2. UV menggunakan lampu deuterium atau disebut juga heavi hidrogen
  3. VIS menggunakan lampu tungsten yang sering disebut lampu wolfram
  4. UV-VIS menggunan photodiode yang telah dilengkapi monokromator
  5. Monokromator berfungsi sebagai penyeleksi panjang gelombang yaitu mengubah cahaya yang berasal dari sumber sinar polikromatis menjadi cahaya monokromatis. Jenis monokromator yang saat ini banyak digunakan adalah gratting atau lensa prisma dan filter optik.

Jika digunakan grating maka cahaya akan dirubah menjadi spektrum cahaya. Sedangkan filter optik berupa lensa berwarna sehingga cahaya yang diteruskan sesuai dengan warna lensa yang dikenai cahaya. Ada banyak lensa warna dalam satu alat yang digunakan sesuai dengan jenis pemeriksaan.

Pada gambar 2 disebut sebagai pendispersi atau penyebar cahaya. Dengan adanya pendispersi hanya satu jenis cahaya atau cahaya dengan panjang gelombang tunggal yang mengenai sel sampel. Pada gambar 2 hanya cahaya hijau yang melewati pintu keluar. Proses dispersi atau penyebaran cahaya seperti yang tertera pada gambar 3.

3Gambar 3 Proses dispersi

           (sumber: Wiryawan, 2008)

 Fungsi masing-masing bagian:

  1. Sel sampel berfungsi sebagai tempat meletakan sampel.

UV, VIS dan UV-VIS menggunakan kuvet sebagai tempat sampel. Kuvet biasanya terbuat dari kuarsa atau gelas, namun kuvet dari kuarsa yang terbuat dari silika memiliki kualitas yang lebih baik. Hal ini disebabkan yang terbuat dari kaca dan plastik dapat menyerap UV sehingga penggunaannya hanya pada spektrofotometer sinar tampak (VIS). Kuvet biasanya berbentuk persegi panjang dengan lebar 1 cm.

  1. Detektor berfungsi menangkap cahaya yang diteruskan dari sampel dan mengubahnya menjadi arus listrik. Syarat-syarat sebuah detektor:
  • Kepekaan yang tinggi
  • Perbandingan isyarat atau signal dengan bising tinggi
  • Respon konstan pada berbagai panjang gelombang
  • Waktu respon cepat dan signal minimum tanpa radiasi
  • Signal listrik yang dihasilkan harus sebanding dengan tenaga radiasi.
  1. Read out merupakan suatu sistem baca yang menangkap besarnya isyarat listrik yang berasal dari detektor.
  1. Proses Absorbsi Cahaya pada Spectrofotometri

Ketika cahaya dengan panjang berbagai panjang gelombang (cahaya polikromatis) mengenai suatu zat, maka cahaya dengan panjang gelombang tertentu saja yang akan diserap. Di dalam suatu molekul yang memegang peranan penting adalah elektron valensi dari setiap atom yang ada hingga terbentuk suatu materi. Elektron-elektron yang dimiliki oleh suatu molekul dapat berpindah (eksitasi), berputar (rotasi) dan bergetar (vibrasi) jika dikenai suatu energi.

Jika zat menyerap cahaya tampak dan UV maka akan terjadi perpindahan elektron dari keadaan dasar menuju ke keadaan tereksitasi. Perpindahan elektron ini disebut transisi elektronik. Apabila cahaya yang diserap adalah cahaya inframerah maka elektron yang ada dalam atom atau elektron ikatan pada suatu molekul dapat hanya akan bergetar (vibrasi). Sedangkan gerakan berputar elektron terjadi pada energi yang lebih rendah lagi misalnya pada gelombang radio.

Atas dasar inilah Spectrofotometri dirancang untuk mengukur konsentrasi suatu zat yang ada dalam suatu sampel. Dimana zat yang ada dalam sel sampel disinari dengan cahaya yang memiliki panjang gelombang tertentu. Ketika cahaya mengenai sampel sebagian akan diserap, sebagian akan dihamburkan dan sebagian lagi akan diteruskan.

Pada Spectrofotometri, cahaya datang atau cahaya masuk atau cahaya yang mengenai permukaan zat dan cahaya setelah melewati zat tidak dapat diukur, yang dapat diukur adalah It/I0 atau I0/I(perbandingan cahaya datang dengan cahaya setelah melewati materi (sampel)). Proses penyerapan cahaya oleh suatu zat dapat digambarkan sebagai berikut.