Etiket Arşivleri: experiment

Laboratory‎ > ‎Immobilization


In this experiment we determined the rate of enzyme activity using free and immobilized enzyme. And our aim in this experiment, notice the difference between them.


Distilled water, Buffer solution, Tube, Pipette, Oil, Alcohol, Water bath, Beaker, Enzyme (Candida rugosa lipase)


Immobilized enzyme: 0.012 g Immobilized enzyme (Candida rugosa lipase) was taken and 1 ml. olive oil was added. Then 3 ml. 25 mM KP buffer was added. This solution was placed water bath at 37 C for 30 min. After immobilized enzyme had been centrifuged, supernatant was taken. And 5 ml. alcohol (ethanol) and a few drops phenolphthalein were added. The time was recorded which is taken from the centrifuged to alcohol added. After that it was titrated with 0.05 M KOH. Finally titrated volume was recorded. Free enzyme: 0.2 ml. free enzyme was weighted then 1 ml .olive oil and 3 ml. 25 mM KP buffer were added. It was placed water bath at 37 C for 30 min. After that 5 ml ethanol and a few drops phenolphthalein were added and titrated with 0.05 M. KOH. Finally titrated volume was recorded.

Laboratory‎ > ‎Km v1

Purpose: The aim of this experiment is determining Km by using three methods which are Michaelis Menten equation, Eadie Hofstee and Lineweaver Burk plot.

Materials: Double beam spectrophotometer, test tubes, pipettes

Enzyme solution: 10 mg/ml enzyme in distilled water

Substrate solution: p-Nitrophenyl acetate in acetonitrile

Buffer: 200 mM potassium phosphate buffer, pH 7.4

Procedure: First of all, according to table below the reagents was diluted into spectrophotometer cell at each substrate concentration for each step in the table. And then the change in absorbance with time was followed by a double beam spectrophotometer for each substrate concentration in the table. However time versus absorbance graph for each substrate concentration was printed by spectrophotometer. From this graph initial rate versus substrate concentration was found. And finally from obtained data mK methods. value of the enzyme substrate reaction values was found by using three

Laboratory‎ > ‎Hydrocarbons


The aim of this experiment is classified of the hydrocarbon and learn the test which are aromatic bromation, permanganate test, sulfuric acid test and nitration of aromatic hydrocarbon. Hydrocarbon , any organic compound composed solely of the elements hydrogen and carbon. The hydrocarbons differ both in the total number of carbon and hydrogen atoms in their molecules and in the proportion of hydrogen to carbon. The hydrocarbons can be divided into various homologous series. Each member of such a series shows a definite relationship in its structural formula to the members preceding and following it, and there is generally some regularity in changes in physical properties of successive members of a series.The alkanes are a homologous series of saturated aliphatic hydrocarbons. The first and simplest member of this series is methane, CH4; the series is sometimes called the methane series. Each successive member of a homologous series of hydrocarbons has one more carbon and two more hydrogen atoms in its molecule than the preceding member. The second alkane is ethane, C2H6, and the third is propane, C3H8. Alkanes have the general formula CnH2n+2 (where n is an integer greater than or equal to 1).  Generally, hydrocarbons of low molecular weight, e.g., methane, ethane, and propane, are gases; those of intermediate molecular weight, e.g., hexane, heptane, and octane, are liquids; and those of high molecular weight, e.g., eicosane (C20H42) and polyethylene, are solids. Paraffin is a mixture of high-molecular-weight alkanes; the alkanes are sometimes called the paraffin series. Other homologous series of hydrocarbons include the alkenes and the alkynes. The various alkyl derivatives of benzeneare sometimes referred to as the benzene series. Many common natural substances, e.g., natural gas, petroleum, and asphalt, are complex mixtures of hydrocarbons. The coal tar obtained from coal by coking is also a mixture of hydrocarbons. Natural gas, petroleum, and coal tar are important sources of many hydrocarbons. These complex mixtures can be refined into simpler mixtures or pure substances by fractional distillation. During the refining of petroleum, one kind of hydrocarbon is often converted to another, more useful kind by cracking. Useful hydrocarbon mixtures include cooking gas, gasoline, naphtha, benzine, kerosene, paraffin, and lubricating oils. Many hydrocarbons are useful as fuels; they burn in air to form carbon dioxide and water.  The hydrocarbons differ in chemical activity. The alkanes are unaffected by many common reagents, while the alkenes and alkynes are much more reactive, as a result of the presence of unsaturation (i.e., a carbon-carbon double or triple bond) in their molecules. Many important compounds are derived from hydrocarbons, either by substitution or replacement by some other chemical group or element of one or more of the hydrogen atoms of the hydrocarbon molecule, or by the addition of some element or group to a double or triple bond (in an unsaturated hydrocarbon). Such derivatives include alcohols, aldehydes, ethers, carboxylic acids, and halocarbons.


Test tubes, water bath, thermometer, pipette, benzene, bromine solution, iron fillings, potassium permanganate (KMnO4), acetone, paraffin, H2SO4, HNO3, distilled water.


Aromatic Bromation:

1 mL of benzene was placed into two seperate test tubes.2-3 drops of bromine solutions was added in each test tubes.A few iron fillings was added one of the benzene samples.Both tubes was placed in a warm-bath at 50°C for 30 minutes.

Permanganate Test:

0.5 mL of benzene was added to 3 mL of KMnO4 solution and it was shaked well.Then 0.1 g solid paraffin was dissolved in 1 mL alcohol free acetone in another test tube.Then permanganate solution was added drop by drop to the second solution.

Sulfuric Acid Test:

0.5 mL benzene was added to 3 mL of concentrated H2SO4 and this solution was shaked gently.

Nitration of aromatic Hydrocarbons:

Mixture of 1 mL HNO3 concentrated with two drops of concentrated H2SO4 was added to 1 mL of the benzene.solution was heated at 50°C of 15 minutes.And then 5 mL of water was poured into the solution.


For the first test aomatic bromation,in each of the two test tube color change was occured.But test tube which is Fe in color change more rapidly.

For the permanaganate test, in first test tube two layer were occured.In second test tube two layer were occured to.Bottom layer’s color was dark,top layer’ color was light. For sulfuric acid test, hydrocarbon which is benzene didn’t dissolved in cocentrated H2SO4.Heat is evolved.

For nitration of aromatic hydrocarbons, two layers were occured.Heavy oil was the top layer.


In this experiment benzene and paraffin were used as hydrocarbon. Benzene, also known as benzol, is an organic chemical compound with the formula C6H6. It is sometimes abbreviated PhH. Benzene is a colorless and flammable liquid with a sweet smell and a relatively high melting point. It is carcinogenic and its use as additive in gasoline is now limited, but it is an important industrial solvent and precursor in the production of drugs, plastics, synthetic rubber, and dyes. Benzene is a natural constituent of crude oil, but it is usually synthesized from other compounds present in petroleum. Benzene is an aromatic hydrocarbon and the second [n]-annulene ([6]).

Paraffin is a common name for a group of alkane hydrocarbons with the general formula CnH2n+2, where n is greater than about 20, discovered by Carl Reichenbach. It is distinct from the fuel known in Britain as paraffin oil or just paraffin, which is called kerosene in American English. Usage of the term varies in other countries, leading to confusion about which substance is being referred to. The solid forms of paraffin are called paraffin wax. Paraffin is also a technical name for an alkane in general, but in most cases it refers specifically to a linear, or normal alkane, while branched, or isoalkane are also called isoparaffins. The name is derived from the Latin parum (= barely) + affinis with the meaning here of “lacking affinity”, or “lacking reactivity”).

            In first test Fe was used as Lewis acid and addition of Lewis acid does cause a facile reaction for the generation of a positive bromine.Permanganate was used as oxidizing agent in second test.In nitration of aromatic hydrocarbon catalyst which is water was used.

‎Laboratory‎ > ‎Melting Point Determinations


The purpose of experiment is melting point determinations of any substances and effect of the impurities on the melting point.


First we will select the a known substances and determine its melting point.Then  we will repeat the melting point determination of the knownsubstance until good results are obtained.We will contaminate the known substance with a foreign substance.And then we will determine its melting point and observe  the effect that contamination has on the sharpness of a melting point.Finally we will obtain an unknown substance and determine its melting point and we will record the results.


1.      There are four aparatus used to measure  the melting points of organic substances.They are melting point bath,long-necked flask,thiele tube and electrically heated melting point.

Ethyl alcohol,acetone,sugar and salt are impurities substance.A soluble impurity contributes to the total vapour pressure,thereforelowering the partial vapour pressure required of the pure substance in the melt and thus lowers the temperature necessary for melting.Addition of more impurity will produce corresponding lowering in the partial vapour pressure of the pure substance and hence ,lowering of the melting point.


Determination of the temperature at which the solid and liquid phases of a substance are in equilibrium is tedious and time consuming; it is also quite difficult with a small amount of sample. Thus, in practice, most melting points are determined as capillary melting points, which can be done quickly with a small amount of sample. A capillary melting point is defined as the temperature range over which a small amount of solid in a thin walled capillary tube first visibly softens (first drop of liquid) and then completely liquefies. Melting points recorded in the chemical journals are capillary melting points unless otherwise stated.

A solid is said to melt sharplyif the melting point range is 0.5 – 1.0 deg. C. A pure solid will generally melt sharply because the forces of attraction between its particles are the same. However, the presence of a foreign particle in a crystal lattice interrupts its uniform structure and the forces of attraction are weakened.

An impure solid melts at a lower temperature and over a wider range. Thus, a solid’s melting point is useful not only as an aid in identification but also as an indication of purity.

We found the melting point of the naphtallin was 82 deg. C.and the range of the naphtallin was 82-86 deg.C.Then we did the experiment with unknown substances and the value of the its melting point was 188 deg.C.We found its melting range 188-192 deg. C.And then we predicted that the substances was succinic acid.Because its melting range was 187-192 deg.C.Our results was a little different from these values.These differences may be source of the experimental error such that we may read the thermometer wrong.


1-We use the capillary tube for determining the melting point of the unknown substances.We put the unknown substances in the capillary tube and then we hold the capillary inside the thiele tube.Then we can determine the melting point.The capillary tube must have thin wall and small diameter so temperature is the same value capillary and thiele tube.And we determine the correct point.

2-The instrument is superior to hot stage for rapid work,since the inner tube and thermometer can be quickly transfered to a cold.Thiele tube for subsequent determinations,where as a hot stage is so throughly insulated as to retain its heat for a long period of time.This is the advantages of the this method.Another advantage is correct result is obtained with using thiele tube but experiment with this method take very long time.This is the disadvantages of the method.

Laboratory‎ > Purification of Organic Compounds


In this experiment distillation which is a seperation and purification method of compounds was used. Distillation is based on the fact that the vapour of a boiling mixture will be richer in the components that have lower boiling points.Therefore, when this vapour is cooled and condensed, the condensate will contain more volatile components. At the same time, the original mixture will contain more of the less volatile material. Distillation columns are designed to achieve this separation efficiently.  Although many people have a fair idea what “distillation” means, the important aspects that seem to be missed from the manufacturing point of view are that:

– distillation is the most common separation technique    – it consumes enormous amounts of energy, both in terms of cooling and heating requirements   -it can contribute to more than 50% of plant operating costs

The best way to reduce operating costs of existing units, is to improve their efficiency and operation via process optimisation and control. To achieve this improvement, a thorough understanding of distillation principles and how distillation systems are designed is essential.

Simple distillation is good for separating relatively low-boiling liquids (less than 150 degrees C) from nonvolatile solids or other liquids with significantly different boiling points.  A 25 degree difference is generally separable from solvent.

Higher boiling solvents can be distilled if pressure over the distillation system is reduced.  This method is called vacuum distillation.

Steam distillation is a method for separating water-insoluble or slightly-soluble substances from water mixtures.  This is used frequently for isolating flavor or fragrance oils from natural products (lavender, mint, etc.).

Some mixtures that seem like they should distill well just won’t.  Those liquids that don’t behave like they ought to (according to Raoult’s Law that describes vapor pressures in mixtures), that have a constant composition and a constant boiling point as they distill, are called azeotropes.  95% ethanol behaves this way.  Most mixtures will undergo composition changes as they distill, so the temperature of the distilling vapor will change with time.  Such is not true for an azeotrope.  The temperature you record during the distillation of one of these special mixtures remains constant.


Acetone , water, simple distillation mechanism, beaker, steam distillation mechanism, coffee, thermometer.


First simple distillation was done.Simple distillation mechanism was installed.Thirty five mL acetone and 35 mL water were placed in distillation flask.Then the mixture was heated.The thermometer was not allowed to show 100°C.Then at 56°C acetone boiled.And 32 mL acetone was collected in the graduated cylinder.

Secondly steam distillation mechanism was set up.Our sample is coffee.Water was heated and then a liquid which smell like coffee was collected in beaker.


1- Fractional distillation is used to separate mixtures of miscible liquids, such as ethanol and water. The process depends on the components of the mixture having different boiling points. The liquid is heated so that it turns into a gas. The vapours pass up a fractionating column where they are gradually cooled. As each of the components of the mixture cools to its boiling point, it turns back into a liquid. The different components of the mixture condense at different levels in the fractionating column and thus may be separated

2-At the boiling point of such a mixture,both components will contribute some molecules to the vapor.The more volatile component,because it is more easily vaporized,will have a larger fraction of its molecules in the vapor state than will less volatile component.When this vapor is condensed into another container,the resulting liquid (the distillate) will be rich in the more volatile component then was the original mixture.As distillation continiues,the boiling point rises until,finally,the boiling point of the less volatile liquid is reached.The first portion of the distillate is has the greater concentration of the more volatile component in the distillate increases.Thus,the more volatile liquid in the mixture can be obtained by collecting only the first portion of the distillate.

3-The packed column,usually glass beads,helices or fingers,gives a large surface area for contact of the ascending vapours and descending liquid.After the first fraction has distilled,the temperature will rise and the rate of distillation will slow.This is an intermediate fraction containing a little of both component of the mixture.Finally,the temperature will become constant and the pure higher boiling compound will distill.


During the distillation, the boiling point (temperature) of the liquid can be observed. Once the boiling point has been obtained, comparison of this number with literature values can help establish the identity of the unknown liquid.The central premise of distillation is that a mixture of liquids can be separated from each other by taking advantage of differences in boiling points between the compounds in the mixture. In order to do this, the components of the mixture are slowly vaporized, condensed, and collected.

Since the components of the original mixture presumably have different boiling points they vaporize at different temperatures, and consequently, are separated by collecting fractions boiling at different temperatures. Normally, if the impurities and desired compounds have boiling point differences of greater than 50°C, the separation is easily accomplished using the simplest of distillation equipment.At first glance, distillation seems like an ideal method, certainly a lot easier than recrystallization. However, it does have its problems. While the boiling points of pure compounds are fairly sharp, generally within a range of 1-2°C, they are not as sensitive to impurities as melting points. In fact, a distillate containing impurities of several percent will not usually have a boiling point significantly different than the pure compound. Constant boiling mixtures, call azeotropes are also possible and may appear to be pure compounds when in fact they are mixtures of compounds.

Some organic compounds decompose with excessive heat. Therefore traditional distillation is not always the best method for separating liquids. In some cases, steam distillation can be used to separate high boiling components from liquid and even solid mixtures. In this experiment, you will be using steam distillation to isolate the essential oils found in spices bought from the grocery store. These spices are complex mixtures of chemicals. The goal is to collect the essential oil from your assigned spice without destroying the chemical nature and physical properties of the compounds. You will use gas chromatography to identify the major components in the essential oil that you separate from your spice. You will be assigned a spice the week before this lab begins, if you have any special requests for spices please let your instrcutor know at that time.The fundamental principle that lies behind distillation is that the composition of a liquid mixture (ie two different liquids mixed together) is different from the composition of its vapour mixture, due to differences in volatility of the liquids in the mixture.

The fundamental principle that lies behind distillation is that the composition of a liquid mixture (ie two different liquids mixed together) is different from the composition of its vapour mixture, due to differences in volatility of the liquids in the mixture. If you mix two liquids (one boils at 100°C, the other at 50°C) then the vapour above the liquid will have much more of the 50°C substance than the 100°C substance. If you then remove the vapour and cool it, so that it condenses, then the new liquid mixture will have much more 50°C substance than the 100°C substance. If you start again heating the new liquid condensed from the vapour (at a slightly lower temperature because it has more of the substance that boils at a lower temperature), then the vapour will again have even more 50°C substance than the 100°C substance, and so on. If you do this enough times, then you can get all of the 50°C substance separate from the 100°C substance. You can get this to happen without having to remove the vapour each time, and you then have continuous distillation, and there is a temperature gradient up the column from hot at the bottom to cool at the top.

Laboratory‎ > ‎Steam Distillation


In this experiment steam distillation method was used.


Distilled water, organic matter (pieces of mint, lemon and apple). Erlenmeyer flask, clamp holder, delivery tube, distillation adapter, water-cooled condenser, Florence flask and Bunsen condenser clamp.


In this experiment, the pieces of lemon were used. This organic compound was distilled with steam distillation. Steam distillation is an ingenious technique for the separation of slightly volatile water insoluble substances from nonvolatile materials. In a mixture of two completely immiscible liquids, each liquid exerts its own characteristic vapor pressure independently of the other. If the water insoluble phase in a steam distillation contains two components, the two different phases (water and organic) distill according to the principle of steam distillation. In other words the molar ratio of the two phases in the distillate is the same as that of the vapor pressures of the two phases.

The organic compounds were not completely insoluble in water at the distillation temperature.

HPLC: Separation And Quantification Of Components In Diet Soft Drinks


Separation And Quantification

Of Components In Diet Soft Drinks


1.      Principles of Instrumental Analysis, 5th Edition, Douglas Skoog, F. James Holler, Timothy Nieman, Saunders College Publishing, Philadelphia, 1998.

2.      “The Analysis of Artificial Sweeteners and Additives in Beverages by HPLC,” Journal of Chemical Education, vol. 68(8), August 1991, p A195-A200.


The purpose of this experiment is to quantify the caffeine content of a diet cola sample using high performance liquid chromatography (HPLC).  In order to quantify the caffeine, it must be isolated from the other components in the mixture.  In this experiment, you will determine a set of HPLC conditions suitable for separating caffeine, benzoic acid, and aspartame and then quantify the caffeine content of your cola sample using a standard calibration curve.  At the end of this experiment you should understand the mechanisms by which components in a mixture are separated, identified, and quantified using HPLC and understand how to vary experimental parameters to optimize a separation.


The fundamentals of chromatographic separations and a detailed discussion of the application of HPLC are covered in reference 1 (chapters 26 and 28).  The following discussion summarizes important concepts from these chapters, but the student is encouraged to read the full text.

Introduction to HPLC and Instrument Components.

High performance liquid chromatography (HPLC) is an important analytical tool for separating and quantifying components in complex liquid mixtures.  By choosing the appropriate equipment (i.e. column and detector), this method is applicable to samples with components ranging from small organic and inorganic molecules and ions to polymers and proteins with high molecular weights.  The various types of HPLC and their characteristics are summarized in the table below.  In this experiment, we will use reversed-phase partition chromatography.

Table 1.  Various Types and Applications of HPLC 







non-polar to somewhat polar

100 – 104

silica or alumina

non-polar to polar

Partition (reversed-phase)

non-polar to somewhat polar

100 – 104

non-polar liquid adsorbed or chemically bonded to the packing material

relatively polar

Partition (normal-phase)

somewhat polar to highly polar

100 – 104

highly polar liquid adsorbed or chemically bonded to the packing material

relatively non-polar

Ion Exchange

highly polar to ionic

100 – 104

ion-exchange resins made of insoluble, high-molecular weight solids functionalized typically with sulfonic acid (cationic exchange) or amine (anionic exchange) groups

aqueous buffers with added organic solvents to moderate solvent strength


non-polar to ionic

103 – 106

small, porous, silica or polymeric particles

polar to non-polar

Figure 1.  shows the components of our Hewlett-Packard Model 1100 HPLC.  The system consists of:

·         reservoirs to hold the solvents used to make up the mobile phase

·         a solvent degasser to prevent bubbles in the mobile phase

·         a programmable quaternary pump that mixes the solvents in the prescribed ratios and pumps them through the column and past the detector

·         a column compartment that houses and thermostats the HPLC column

(in our case a ZORBAX, reversed-phase C18 column; dimensions 4.6mm x 15 cm)

·         an autosampler that draws prescribed volumes from sample vials and injects them onto the column

·         a diode array detector that monitors the entire UV-vis spectrum of the column effluent at regular intervals

Control of the above components and data acquisition and analysis are performed on a personal computer. 

Figure 1.  HPLC 1100 Instrument Components

Optimization of Resolution and Column Performance.

The goal of any HPLC experiment is to achieve the desired separation in the shortest possible time.  Time is critical because “time is money” and because as we’ll see, the more time the sample spends on the column, the more the bands containing the components spread, resulting in reduced resolution.  Optimization of the experiment thus usually involves manipulation of column and mobile phase parameters to alter the relative migration rates of the components in the mixture and to reduce zone broadening.  These can generally be optimized fairly independently.

Migration Rates

The length of time it takes for a given component/solute to travel through the column and be detected is determined by the flow rate of the mobile phase, m, and the partitioning of the solute between the mobile and stationary phases.  Since the solute molecules can only travel when they are dissolved in the mobile phase, the greater their concentration in the mobile phase, the faster they will elute.  The partition coefficient, K, is defined in equation 1

where CS is the concentration of the solute dissolved in or adsorbed to the stationary phase, and CM is the concentration of the solute in the mobile phase. 

The quantities CS and CM, however, are rarely determined in chromatographic experiments.  Instead, a quantity called the retention factor, k’, is determined.  The retention factor for a component A is defined as

where tR is the retention time of component A and tM is the retention time of an unretained species (also called the dead time).  The average rate of linear migration of component A is related to both the flow rate of the mobile phase and the retention factor. 

The retention factors should normally lie in a range of 2-5, but for complex mixtures a larger range may be required to separate all the components.  The value of the retention factor for a given component depends on the chemical identity of the component and the following experimental variables:

·         mobile phase flow rate

·         mobile phase composition

·         column temperature

·         column composition

Zone Broadening

The extent to which the component bands spread as they travel down the column affects the efficiency of the separation.  The theoretical plate height, H, is defined in equation 4 and is based on a Gaussian analysis of the peak width, s, as it exits the column at point L.


and W is the width of the peak at the base.  The data analysis program on our HPLC actually reports the width at half maximum, W1/2, for each peak rather than the width at the base.  Assuming a Gaussian peak shape,


The number of theoretical plates in the column, N, is

Efficient columns have small H and large N for a given component.  The theoretical plate height is affected by the following experimental parameters:

·         mobile phase flow rate

·         diffusion coefficient of the solute in the mobile phase

·         diffusion coefficient in the stationary phase (depends on temperature and viscosity)

·         retention factor

·         diameter of the particles packing the column

·         thickness of the liquid coating on the stationary phase


The resolution of two adjacent peaks, RS, is determined by their separation and their widths.

In other words, RS depends on both migration rates and zone broadening.  A resolution of 1.5 means that the overlap of the peaks is about 0.3%, so conditions should be optimized to achieve at least this resolution if possible. 

In this experiment, you will adjust only the composition of the mobile phase to optimize the retention factors and resolution.  We will not attempt to optimize the zone broadening independently by changing the column or the flow rate.

Components in Diet Soft Drinks

            The ingredient list for most diet soft-drinks includes caffeine, benzoic acid, and aspartame (Nutrasweet®).  The structures of these compounds are shown below along with their UV-vis spectra. 



MW = 194.19

pKa = 10.4

Benzoic Acid




Provided by instructor:

mobile phase  –           HPLC-grade methanol

                                   20 mM phosphate buffer, pH 3

standard samples  –     mixture containing caffeine, aspartame, benzoic acid and uracil

                              –     individual samples of each of the above components        

Student prepared:

diet cola sample –        degassed for 20 minutes with air stream followed by filtration with 0.22mm syringe filter

caffeine standards –    100% = 0.045g/250 mL water

                                    85% dilution




                                    (and additional dilutions as necessary to bracket cola sample)


Model 1100 Hewlett-Packard HPLC system

ZORBAX reversed-phase bonded C18 HPLC column (4.6 mm x 15 cm)

250mL volumetric flask

40-200 mL automatic pipet

200-1000 mL automatic pipet

1mL HPLC vials with caps

vial crimper

analytical balance

Notes on Use of HPLC:

·         Your instructor will show you how to use the equipment and the software.  A primer guide/refresher is also found in the appendix.

·         At the beginning of the day, first open the valve with the black knob on the front of the pump manifold.  This sends the mobile phase to waste instead of the column.  Allow the pump to run for about 10 minutes at 5 mL/min to purge the lines of any air bubbles.

·         At the end of the day, run an 80% water/20% methanol mixture through the column at 1 mL/min for 10 minutes followed by 100% methanol for 10 minutes.  This should flush the column of any potential salt forming materials and stores the column in a compatible solvent.

Sequence of Analysis:

1.      Determine optimum conditions for separation of caffeine, benzoic acid and aspartame using the standard sample provided.  Start with a 75% buffer/25% methanol mobile phase mixture at 1 mL/min, 1mL injection volumes, and a 20 minute run time.  Uracil has been added to the standard mixture to provide a dead time marker (i.e. uracil is unretained).  The diode array detector can monitor several wavelengths at once as well as record the entire spectrum at specified intervals.  Use the UV-vis spectra of the components shown above to choose appropriate wavelength(s) to monitor the chromatograms.  Vary the ratio of buffer to methanol to achieve an acceptable separation and finally adjust the run time to end after the last component exits the column.  Use these data to calculate the retention factors for each component, the resolution between each pair of adjacent peaks, and the values for H and N for caffeine under these conditions.

2.      Using the conditions determined above and the pure samples of each component provided, determine the retention time of each component (i.e. identify the peaks).

3.      Using the column conditions determined above and 5mL injection volumes, run three samples of your most concentrated caffeine standard to determine the precision of the injections.  Then run the remaining caffeine standards and the diet cola sample to determine the concentration of caffeine in your sample using a calibration curve.  Check the full spectrum of each peak against spectra of the pure components to verify that the peaks represent isolated components.

The chemstation control software is accessed via the start menu, programs, HP Chem Stations, and then instrument 1 online.  All of the components are controlled via the run control window shown below by accessing the instrument sub-menus for each of the components.  Notice also that the status of each component is indicated on the run control screen.

The parameters are set in the various “set” menus.  The “more” menus contain control sub-menus that allow you to actually turn these components on.

You can either run individual samples one at a time using the Run Method command under the Run Control menu, or run a sequence of samples using the Sequence menu and it’s various sub-menus.  If you choose the run method option, use the Sample Info menu under the Run Control menu to tell the computer where to store your files and what to name each run.  If you use the sequence mode, you can enter this information in the sequence parameters sub-menu.

Once the data has been collected, use the View menu to see the report containing the chromatograms at each monitored wavelength and the integrated peak areas etc.  From here you can print the report as well as view and print full spectra at any peak.




NAME ________________________________                          DATE ______________


Mass caffeine   _____________

Dilution volumes:       standard    /      water

                       A:        _______  /  _________

                       B:        _______  /  _________

                       C:        _______  /  _________

                       D:        _______  /  _________

                       E:        _______  /  _________

Optimization of Conditions:

% buffer                     ____________

% methanol                ____________

flow rate                     ____________

column pressure         ____________


Retention Time (min)

Retention Factor (k’)














Benzoic Acid




Rcaffeine,aspartame  _______________

Hcaffeine  ___________________

Ncaffeine  ___________________

Quantitation of Caffeine:

wavelength chosen for quantitation  __________________


[caffeine] (mg/L)

Peak Area

diet cola    

avg. peak area for A  ____________________  95% CIm  __________________

slope   ___________________

std. dev. slope  _________________        95%CI slope   ____________________

intercept  ___________________

std. dev. intercept  _______________      95%CI intercept   __________________

R2  ________________

covariance  ___________________

[caffeine]diet cola  _____________________  95%CI  ____________________

Discussion Questions:

Discuss how the choice of monitored wavelength affects the sensitivity of the analysis.  Would you choose the same wavelength to quantify benzoic acid or aspartame?

Based on the pKa’s of the components in our samples, why do you think a mobile phase buffer of pH 3 was chosen? 

If significant zone broadening had resulted in unsatisfactory resolution in our experiment, what might we have changed to reduce its affect?  Comment on the feasibility of this.

Compare the precision obtained for the three injections of the same sample to the precision of your calibration curve.  Which limits the precision of your unknown concentration?  Would using an internal standard be justified?

Show sample calculations and attach all relevant chromatograms and spreadsheet printouts.

Laboratory‎ > ‎DSC (Differential Scanning Calorimeter) and IR (Infrared Absorption Spectroscopy)


In this experiment we have used DSC (differential scanning calorimeter) and IR(infrared absorption spectroscopy).Main principle of working DSC is to measure enthalpy differences of two substances when they are heated. We can measure the amount of energy released or absorbed by the sample by using DSC. By using SC we can determine the heat capacities of substances and find the melting or crystallization temperatures of substances. This information’s gives us characteristic properties of substances and their structures.


Differential scanning calorimetry measures the amount of energy (heat) absorbed or released by a sample as it is heated, cooled, or held at a constant temperature. Typical applications include determination of melting point temperature and the heat of melting; measurement of the glass transition temperature; curing and crystallization studies; and identification of phase transformations.ıt is a technique we use to study what happens to polymers when they’re heated. We use it to study what we call the thermal transitions of a polymer. And what are thermal transitions? They’re the changes that take place in a polymer when you heat it. The melting of a crystalline polymer is one example. The glass transition is also a thermal transition.