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CH 4200, Fall 2004 Prof.

Greenlief

Chromatographic Instrumentation: Gas Chromatography I. Instrument Setup


I.A. General Configuration of the Instruments Two GCs are located in room 102, an HP 5890 (instrument I), and an HP 5980 series II (instrument II). Instrument I has a single capillary column and a flame ionization detector (FID). It uses helium as a carrier gas and as the make-up gas, and a mixture of H2 and air to support combustion in the FID. The make-up gas carries the small volume of gas exiting the column to the detector, providing the optimal gas pressure in the detector. Instrument II has two capillary columns. Column A has a FID, and column B has an electron capture detector (ECD). Column A uses helium as the carrier and He as the make-up gas. Column B uses nitrogen as carrier and nitrogen as the make-up gas. Nitrogen is used as the make-up gas because the ECD requires a gas that is not electronegative. Each instrument is connected to a dedicated integrator readout device. I.B. Gases Four gases are required for the two GCs: helium, nitrogen, hydrogen and air. These are supplied by high pressure cylinders located to the left of the instruments. Gas hookups are as follows: Instrument II injector A He injector B N2 Aux C He (column A make-up) Aux D N2 (column B make-up) Aux E Air (for FID) Aux F H2 (for FID) Instrument I Injector A Aux Make-up gas FID Combustion mixture

He He H2 and Air

The table below gives pressure settings that are appropriate for instrumental operation. Regulated Output at Cylinder 40 psi 30 psi 16 psi 40 psi Typical Column Head Pressure 5-11 psi 11-15 psi

He N2 H2 Air

Full cylinders are pressurized to 2000psi or more. The cylinder pressure is indicated on the righthand regulator guage, and the regulated output pressure is indicated on the lefthand gauge. (See figure below) The cylinder is opend to the regulator when the cylinder valve is turned counterclockwise. The handle on the front of the regulator controls the regulated output pressure, and provides relatively low pressure (<80 psi) at the regulator outlet. The outlets of some regulators are fitted with shutoff valves. The cylinder pressure will drop as the cylinder empties, and will drop rapidly when the cylinder is nearly empty. The low pressure regulator setting should not be changed during normal operation. The column head pressure settings

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CH 4200, Fall 2004 Prof. Greenlief

determine the carrier flow rates through the columns. These can be changed within certain ranges in order to optimize the analytical method being used, as described in section I.B.1.
regulated output pressure tank pressure cylinder valve shut-off valve

I.C. Columns The columns currently in use are wall-coated open tubular capillary columns. Column A is a 15 meter column (0.32 mm inside diameter) with a polar cyanopropylsilicone coating. The injector closest to the front of the GC loads a sample onto column A; the effluent from column A goes to detector A, the FID detector. Behind injector A is injector B, which leads to column B, a 30 meter column (0.32 mm inside diameter) with a nonpolar 5% phenylmethylsilicone coating. The effluent from column B goes to detector B, the ECD detector. I.D. Data and Electrical Cables The chromatograph is connected to the integrator via two cables using HP's proprietary INET. These cables carry data and control signals between the GC and the integrator. The only other electrical connections are to the 110V power supply. I.E. Preparation Prior to Running a Sample The following procedure is recommended if the instrument has been left at room temperature for more that a few weeks. Adjust carrier gases so that a flow rate of at least 2 ml/min flows through all columns in the instrument to be used. Set temperatures as listed below and allow the carrier to flow through the column at elevated temperature for at least 24 hours in order to purge the columns. Test the columns using the benchmark tests described below. If excessive noise persists, purge the columns again. To heat each device press the appropriate device button on the keypad. The display will report the current temperature of the device. Enter the desired temperature, then press the ENTER key to set the temperature. Press ON to turn the device on. The instrument will now adjust the temperature to the desired value. Instrument II Detector Temperature Detector A: 250 oC Detector B: 300 oC Injector temperature Both injectors 220 oC Instrument I Detector Temperature 250 oC Injector Temperature 220 oC

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CH 4200, Fall 2004 Prof. Greenlief

Oven Temperature for column bakeout 150-180 oC

Oven Temperature for column bakeout 150-180 oC

I.F. Shutdown for Long Dormant Periods Turn all heated elements off. First press the appropriate keypad button, then press the OFF button. It is not necessary to change the temperature once the OFF button has been pressed. After the oven cools down below 50 oC, turn off the gases at the cylinders. Turn the cylinder valve (not the regulator) clockwise to close the cylinder. II. Instrument Settings and Operation II.A. General Principles In a gas chromatograph, there is a column which is located in an oven. An injector is at one end of the column and a detector is at the other. The injector allows the user to load a sample onto the column via a syringe. An inert carrier gas, usually helium, flows through the column from the injector end to the detector end at all times. Volatile components of the injected sample are vaporized in the injector and are carried through the column to the detector via the carrier gas stream. The column serves as the support for a bonded, thin-film stationary phase. As the volatile components (analytes) of the sample are carried along the column, they interact with the stationary phase. The degree of interaction between analyte and stationary phase influences the time it takes for a particular analyte to come off the column. This time is known as the retention time. A strongly interacting analyte will take longer to pass through the column than a weakly interacting analyte. Column temperature and stationary phase composition greatly influence the retention times in gas chromatography. Choosing a column of an appropriate polarity can aid in separating compounds with similar boiling points. However, temperature, which controls the volatility, can not be ignored. In a fixed temperature or isothermal separation, the lower boiling components of a mixture will elute from the column prior to the higher boiling ones. Thus, isothermal gas chromatography can be used to separate higher boiling components from the lower boiling ones based on their respective retention times. Because of the long retention times for higher boiling components, most modern GC separations use temperature programming in which the column temperature is varied during the analysis. Faster elution of high boiling components which prevents peak broadening and decreases analysis time is realized by increasing the column temperature. Temperature programming provides a good separation of both high and low boiling components. Detector choice is also an important consideration in gas chromatography. The flame ionization detector (FID) is widely used because it responds well to all compounds and has good sensitivity (see, for example, Skoog, et al., Instrumental Analysis, (Harcourt Brace & Co. 1998: Philadelphia) for additional detail). Another common detector is the electron capture detector (ECD) which is especially sensitive to and selective for compounds containing Cl, O, and other electronegative elements. The ECD is useful for environmental analyses of organochlorine pesticides and polychlorinated aromatics. The ECDs selectivity and sensitivity leads to very low detection levels of electronegative elements.

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CH 4200, Fall 2004 Prof. Greenlief

The more commonly used FID detectors ionize analytes via a hydrogen flame placed at the end of the column. The detector monitors the ion current as it passes through. Under these circumstances, most compounds will ionize to some extent, but compounds containing electronegative elements do not ionize very well. Therefore, the FID detector response to electronegative elements is lower than simple hydrocarbons. An ECD detector operates on the inverse principle. It contains an electron source, and the detector monitors the electron current from this source. When a compound containing an electronegative element passes through the detector, the electrons are captured and the current drops, thus explaining why the ECD is highly sensitive to these types of compounds. ECD and FID are complementary detection schemes. II.B. Gases and Flow Rates Initial cylinder pressure settings are described in the Instrument Setup section above. These gases provide service to each injector, column and detector. Each of these devices requires gas flow, and separate valves are provided to control flow rates to each divice. The carrier flow rate through the column is a critical parameter that must be maintained throughout the analysis. This flow rate can be measured directly with a bubble meter by attaching the flow measuring adapter to the FID output and connecting this to the hose on the bubble meter. (This adapter must be removed from the FID output prior to igniting the hydrogen flame!) It can be measured on the ECD by connecting the ECD vent tube on the back of the instrument into the bubble meter tube. The measured flow rates will depend on the temperature settings of the inlet and the oven, and on the length and diameter of the column. The direct measurement is tedious, but it has been found that the column head pressure determines the flow rate on the column. A 2 ml/min flowrate is desirable in most cases, and the head pressures listed below give 2 ml/min under the given conditions. The column flow rate is controlled by adjusting the column head pressure valve on the front left instrument panel. Each injector port has a separate set of control valves. Each of the columns has an inlet with a split injector and a septum purge. The septum purge on each column should be set to 3 ml/min. This can be easily measured directly by attaching the bubble meter to the purge vent on the front left panel. Adjust the purge valve to give 3 ml/min flow. The split vent is used to simplify the injection procedure. Capillary columns have low capacity, and therefore require very small analyte samples. Sometimes the sample volumes are inconveniently small, so the split injector has been designed to discard a fraction of the volatilized sample. The discarded sample flows out of the split vent located on the left front instrument panel. If the column flow rate is 2 ml/min and the split vent flow rate is 6 ml/min, then of the sample is being discarded. This is a split ratio of 3:1. The larger split ratio discards more sample, and therefore affects sensitivity. Measure the split vent flow rate and use the total flow valve to adjust the split ratio. The split ratio is selected by the user to suit a particular analysis and sample. The detectors also require additional gas, and these flow rates must now be adjusted. Two sets of detector flow valves are located on the left front instrument panel. The detector valve are only used to turn the various gases on and off at the detectors, and cannot be used to control flow rates! Both FID and ECD require make-up gas in order to carry the analyte that exits the column into the detection zone. Without the make-up gas, the analyte will linger at the

Chromatographic Instrumentation: GC-4

CH 4200, Fall 2004 Prof. Greenlief

end of the column because the volume of gas that flows through the column is not sufficient to drive it through the relatively large volume of the detector. Makeup gases are turned on and off with the AUX valve on the upper-left instrument panel. The FID makeup gas is He, and its flow rate is adjusted by turning a screw inside of the FID AUX valve. It should be set so that the column + makeup flow rate is 30 ml/min. The ECD makeup gas is N2, and its flow rate is controlled by adjusting the regulated output pressure at the N2 tank. It should be adjusted to 3060 ml/min. It is turned on or off using the ECD AUX valve. The ECD detector also has an anode purge that keeps foreign material from acumulating in the detector. The anode purge is turned on or off with the ANODE PURGE valve on the upperleft front instrument panel. Its flow rate is also controlled by the regulated output pressure. The anode purge and the ECD makup gas flow rates cannot be independently adjusted. The cylinder regulated output should be adjusted to give an anode purge flow rate of 3 ml/min. The ECD makeup gas flow rate can then be checked, and it should be 30-60 ml/min. The FID detector also requires H2 and air to support a flame. On-off valves for each of these gases are located on the upper-left instrument panel. The flow rates are set by varying the regulated output pressures ate the cylinders. The flow rates can be set with the aid of the front panel TIME feature. Press TIME repeatedly until t=0.00 1/t= 0.00 appears. Pressing ENTER once initiates a stopwatch feature. Pressing ENTER again stops the stopwatch. Press CLEAR to reset the stopwatch to zero. Using the buble meter, start the stopwatch when a bubble passes 0, and stop it when it reachs the desired volume mark, given in ml. Use the volume and the time to calculate the flow rate. The following lists give the correct sequence and procedure for adjusting flow rates for the 5890 Series II columns, injectors, and detectors.

FID SYSTEM Component Flowrate column 2 ml/min

Control Valve column head pressure

Comments and conditions Install flowrate measurement adapter tube in end of column. Turn off makeup, H2 and air at front panel. Typical head pressure is 5 psi. this flow rate is typical Install flowrate measurement adapter tube in end of column. Turn off H2 and air at front panel. Turn the makeup gas valve on. The measured flow is makeup + carrier. turn off makeup gas and air. Typical pressure is 16 20 psi. turn off makeup gas and H2. Typical pressure is 40 psi.

septum purge 3 ml/min split vent 6ml/min makeup gas 30 ml/min

purge valve split valve AUX set screw valve

H2 Air

30 ml/min 400 ml/min

H2 regulator Air regulator

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CH 4200, Fall 2004 Prof. Greenlief

ECD SYSTEM Component Flowrate column carrier 2 ml/min

Control Valve column head pressure

Comments and conditions Attach flow meter tube to vent tube on back of instrument. Turn off makeup, and anode purge on front panel. Typical head pressure is 11 psi. this flow rate is typical attach flow meter tube to vent tube on back of instrument. Turn off anode purge at front panel. The measured flow is makeup + carrier. attach flow meter tube to vent tube on back of instrument. Turn off turn off makeup gas on front panel. Typical pressure is 16 20 psi.

septum purge 3 ml/min purge valve split vent 2ml/min split valve makeup gas 30-60 ml/min N2 regulator

anode purge

3 ml/min

N2 regulator

II.C. Temperature Programming The operation of the instruments is controlled by the keypad. You can monitor the GC conditions by pressing the appropriate buttons. For example, if you press OVEN TEMP the display will read out the actual current temperature as well as the temperature to which the oven has been set. To set the oven temperature, you would press OVEN TEMP, the desired temperature on the numeric keypad, and ENTER. Other functions work similarly. The following list describes the more important functions of the instrument, particularly those used to define a temperature program. Other functions are explained in greater detail in the instruments manual. A. Parameters for Temperature Programs INIT VALUE the initial temperature for the program INIT TIME the length of time to hold at the initial temp RATE the rate of temperature ramp in 0C per minute FINAL VALUE the temperature at which the ramp stops FINAL TIME the length of time to remain at the final temperature before cooling back down to INIT VALUE B. Parameters for Detector SIG 1 - Pressing sig 1 once will cause the display to readout the signal level of signal one continuously. Pressing it again will tell the operator which detector has been defined as signal 1. You can change this by selecting A or B and then pressing enter. SIG 2 - The similar to sig 1. (Inoperable on this instrument.) DET - Informs the operator which detector is A and which is B, and allows the operator to turn these ON or OFF.

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CH 4200, Fall 2004 Prof. Greenlief

II.D. Use of the HP 3396 Integrator The GC is equipped with an integrator, a device which measures the intensity of the detector signal, electronically integrates the peak areas, and provides a peak report for the chromatogram at the end of a run. The following instructions describe its operation. NOTE: The integrator only monitors one detector at a time, the detector assigned to SIG 1. You need to instruct the GC to send the correct signal to the integrator. To do this, press SIG 1, then press A if you want to monitor detector A. Press SIG 1 then B to change the monitor detector B. Setting parameters: To set a parameter, press the applicable key and enter the desired value, followed by the ENTER key. ATT2^ value sets the attenuation of the signal output from the GC. Value is from 0 to 13. The relationship between the signal provided by the GC (signalin) and the signal displayed by the integrator (signalout) is as follows: signal in signal out = 2n where n is the attenuation setting. A value of 3 or 4 is usually appropriate. CHT SP value sets the chart speed to value cm/min. 1 cm/min is usually quite adequate, but 2 cm/min may be needed to see resolved but closely eluting peaks. AR REJ value sets the minimum area a peak must have in order to be labeled and listed in the peak table. A value of 5000 is a good starting place. THRSH value sets the minimum height a peak must have in order to be integrated. A value between 1 and 3 is probably reasonable. PK WD value sets the minimum width a peak must have in order to be integrated. Values between 0.01 and 0.05 (inches) are reasonable. Listing parameters: Pressing the LIST key followed by any one of the keys indicated above will display the specified information about the integrators current settings.

Pressing LIST LIST displays the current settings for the above parameters plus a peak capacity (the number of peaks the integrator can store, given the current settings) and the zero level. Pressing LIST METHOD 8 followed by the ENTER key prints the current GC settings, including: temperature program settings injector and detector settings and temperatures signal settings and detector status The printout is close to a page long.
II.E. Running a Sample Proper Injection Technique - follow these instructions carefully for all injections!!!

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CH 4200, Fall 2004 Prof. Greenlief

To start a run, you inject a one microliter (l) sample with the syringe. First, draw about one microliter of air into the syringe, and then several microliters of sample. Expel all but one microliter of the sample. Note that you want a 1 l plug of sample surrounded by air on both sides in the syringe. This way you ensure injection of the entire sample into the column, not just into the needle. See the figure below.

Properly Filled Syringe


air sample air solvent plunger

You are now ready to make your injection. As you insert the needle into the injector septum, you must hold onto the plunger with one hand while directing the needle with the other. If you do not hold the plunger, the head pressure will push it out of the syringe body, along with the sample. You will encounter some resistance when inserting the needle, but if you hold it straight and keep it from bending, you will be able to insert at least 3/4 of its length into the injector. At this point, you depress the plunger with a swift motion, and then press START on the front panel. If the run ends (i.e., all analytes elute) before the programmed stop time, you can end the run by pressing stop.
II.F. Overnight Shutdown Procedure Leave the injectors and detectors at operating temperatures. Adjust the column temperature to 40 oC (if it hasn't returned there automatically). Turn off the H2 and air at the cylinder. Leave the carrier gases ON to protect and purge the columns. II.G. Test Samples

Separation and Detection of Representative Hydrocarbons and Halocarbons In this experiment, you will evaluate the response signal of the ECD detector and the FID detector to two samples. The first contains the following compounds at the concentrations listed below. We will refer to it as the halocarbon mixture chlorobenzene nitrobenzene 1,3-dichlorobenzene 1,2,4-trichlorobenzene 5 mM 0.025 mM 0.01 mM 0.002 mM 0.5628 mg/ml 0.0031 mg/ml 0.0015 mg/ml 0.0004 mg/ml

The ECD should be particularly sensitive to this mixture since all of the compound contain electronegative elements. The second mixture will contain the following compounds. We will refer to it as the hydrocarbon mixture.

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CH 4200, Fall 2004 Prof. Greenlief

toluene p-xylene t-butylbenzene

10 mM 10 mM 10 mM

0.92 mg/ml 1.06 mg/ml 1.34 mg/ml

You will inject each of these mixtures on each of the columns for a total of 4 injections. The TA will provide the retention time for each of these compounds on each column when using the following temperature program. SIG 1 INIT VALUE INIT TIME RATE FINAL VALUE FINAL TIME A when using the FID, B when using the ECD 40 C 3 min 20 C/min 220 C 5 min

INTEGRATOR ATTENUATION: SEE TA In addition to these 4 injections, you will need to inject an air sample, a benzene solvent sample, and a hexane solvent sample on each column in order to characterize the retention time of an unretained species on the column, and to identify the solvent peaks. Make sure to use the same temperature program for each injection.
Report Format

Make a table of retention times, peak widths and peak areas for each mixture on each column for a total of 4 tables. Be sure to include the retention times for air and for the solvent in each of the tables. Also include the peak area of a small impurity peak that can be used to estimate the background level of the signal. Your TA can help you identify such a peak. Label each table with a title such as Hydrocarbon Mixture on Column B with ECD Detector. Then answer the following questions:
Question 1: List in order, from highest to lowest, the sensitivity of each detector to the various compounds as observed on your chromatogram, and discuss these results.

Use units of peak area per mg/ml. Which compounds are detected most sensitively with the ECD? With the FID? Explain this result. What problems arise when you attempt to make this comparison? (Hint: See question 3 in the HPLC section.) Estimate the limit of detection for toluene on the ECD chromatogram and trichlorobenzene on the ECD chromatogram using the following approximations and assumptions: a.) Assume that peak height is proportional to peak area. b.) Assuming that the minimum detectable concentations must give a peak height equal to 3X the average noise Nav. c.) Approximate Nav=Np-p/5, (one fifth of the peak to peak noise). Now estimate the peak-to-peak noise, calculate an average noise, and calculate the height of the minimum detectable peak as 3Nav. Compare this height with the measured height of each analyte peak (the analyte concentration is known) and estimate the limit of detection for each analyte in mg/ml. Use the impurity peak as a benchmark to estimate the area of a peak that has a

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CH 4200, Fall 2004 Prof. Greenlief

height that is approximately equal to the noise level by assuming that the area is proportional to the peak height.
Selectivity Factor Ideally, the components of the sample should be completely separated by the column. In reality this is not always achieved. Unresolved peaks will be seen on your chromatograms. (This problem is minimized by use of the capillary columns.) The amount of peak overlap arises both from the closeness of the peaks and their widths due to their relative retention times. The selectivity factor, , is defined by the expression
= (t r 2 t 0 ) ( t r1 t 0 )

where t0 is obtained from the air peak (retention time to first part of the solvent peak). You may need to inject a syringe full of air to get an accurate measure of this time. is also the ratio of the partition coefficients, K1/K2, of the two compounds giving rise to the peaks. Partition coefficients are a function of the nature of the stationary phase and of any interaction of the sample components with this phase. If the partition coefficients of two components are closely similar, their retention times or volumes also will be closely similar, and their two peaks may be incompletely separated.
Question 2: Calculate the selectivity for toluene and p-xylene in each column. Do they differ? Why do you expect this result? Column Efficiency As noted above, peak overlap is also a function of peak width, which is not considered by . Peak width is a function of the number of theoretical plates and depends on the column itself as well as the operating conditions. The number of theoretical plates, N, is calculated from the retention time and width, w, of a peak as follows:
t t0 N = 16 r w Question 3: Calculate N for the first and last analyte peak in each chromatogram for a total of 8 calculations. Report your results in a table, and then discuss the results. Are they the same for the different columns? For the different mixtures on the same column? Why or why not? Should N change for different components? Explain.
2

Question 4: Calculate the average height of an equivalent theoretical plate (HETP) or plate height (H) for each of the 4 chromatograms using the equation H=L/N, where L is the column length (polar FID column - 15 meters; nonpolar ECD column - 30 meters) and N is the number of theoretical plates. Use the average of the two N values measured for each chromatogram to get the average HETP for the particular mixture on the particular column.

Chromatographic Instrumentation: GC-10

CH 4200, Fall 2004 Prof. Greenlief

Resolution In order to provide a number to express the extent of separation of two adjacent, approximately Gaussian, peaks the resolution, R, of a column is defined as:
2( t r 2 t r 1 ) (w 1 + w 2 ) where the retention time for peak 2 (tr2) is longer than that of peak 1 (tr1), and w1 and w2 are their respective widths. The peaks are considered to be fully resolved when R = 1.5, but for practical purposes R = 1 is usually adequate (approximately 98% separation). R=

Question 5: What is the resolution (R) of the last two analyte peaks in the ECD mixture chromatogram? What is R for the toluene and p-xylene peaks?

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CH 4200, Fall 2004 Prof. Greenlief

Chromatographic Instrumentation: High Performance Liquid Chromatography I. Instrument Setup


I.A. General Configuration of the Instrument The instrument that will be used for the HPLC experiments is a Beckman 126 Pump (dual pump system). The apparatus includes up to eight HPLC solvent reservoirs (which should be placed in the placeholders on the manifold above the HPLC setup), a diode array absorbance detector (Beckman 168), a manual injection port, an autosampler (Beckman 507e) and a C18 reversed-phase column. The first solvent reservoir, marked Pump A, should contain ultra-pure water obtained from the small room off to the side with has the 18 megaohm water purifier. The second solvent reservoir should contain HPLC grade acetonitrile. There should also be an attached computer with networked printer that controls the functioning of the HPLC. I.B. Liquid Carrier Solvents The solvents that are used for the experiments (water and acetonitrile) must be high quality solvents and they must be degassed. In most cases, degassing the solvents via sonication is sufficient. The presence of air bubbles can cause serious problems in the separation/detection. I.C. Pumps Each pump is designed with a pressure sensor, in order to shut down the pump if it exceeds the maximum pressure limit. The default maximum pressure limit set by the computer is 2500 psi, however, limits can be set as high as 6000 psi. For most of the experiments, the maximum pressure was set to 5000 psi.

Each pump is also equipped with a priming port protruding out and perpendicular to the pump heads. This port is used to prime the pumps, or solvent transfer lines, in the case that the pumps have not been active for some time, or a complete change of mobile phase is required.
I.D. Columns The Beckman system is already equipped with a 25 cm C18 reversed-phase column. The stationary phase is an 18-member alkyl chain. The stationary phase is extremely non-polar, thereby retaining nonpolar species in the sample mixture. I.E. Data and Electrical Cables These should be connected and should remain connected. All connections are selfexplanatory, and all wires are labeled as to which port they should be connected to. Further questions should be directed to the manuals for the instrument. I.F. Preparation Prior to Running a Sample Prior to running a sample, the solvents must be flowing properly through the flow tubes prior to the pump head and through the column with no leakage. Column leakage can be eliminated by gently tightening the fittings on each end of the column. CAUTION: DO NOT

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CH 4200, Fall 2004 Prof. Greenlief

OVERTIGHTEN AS IT IS EASY TO SNAP THE FITTINGS. It is imperative to make sure that there are no air bubbles in the lines from the reservoirs to the pump heads. This can be accomplished by using the special syringe supplied with the system which has a long piece of tubing with a ferrule on the end. The procedure is as follows:

Stop the flow on both pumps (this is done in the direct control window of the 32Karat Software package) Open the priming port on Pump A by turning the silver lever to prime line. Insert a disposable syringe in to the priming port Draw out ~1 mL of solvent, and then empty into the solvent exhaust (from the detector). Repeat this until there is no air in the line and very little air in the syringe (although it is nearly impossible to obtain NO AIR in the syringe as a small amount of air seems to reside in the syringe at all times). Turn the priming lever back to operate When no air can be seen in the line, and very little air is left in the syringe, take the syringe and turn it upside down so that the air bubble is at the top, and begin the somewhat frustrating task of trying to evacuate all of the air (it is especially difficult when the air bubble is very small). When there is no air left in the syringe, reopen the priming port, this time turn it to prime pump and insert the syringe again (WITH THE SOLVENT STILL IN IT). Turn on Pump A to approximately 2.0 mL/minute in the Direct Control module in the computer interface. The Pump flow rate can be set by clicking the text box on the pump on the left side of the screen Begin to apply gentle pressure to the plunger of the syringe (VERY GENTLE), and some of the solvent will enter the pump head. You have PRIMED Pump A. Return the priming port to operate To check the Pump A is working properly, turn the Pump Flow Rate to approximately 1.0 mL/minute. If the pressure indicator on screen reports around (or greater than) 1.000 kpsi, then the pump is sufficiently primed. Repeat for Pump B. Once the pumps have been primed, the system is ready for equilibration and finally experiments.

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CH 4200, Fall 2004 Prof. Greenlief

Shutdown for Long Dormant Periods This instrument should not be completely dormant for long periods of time. The alternative is to fill the reservoirs as much as possible as run the pumps at a very low flow rate. This keeps the column conditioned.

II. Instrument Settings and Operation


II.A. General Principles In reversed-phase HPLC, the stationary phase is non-polar, while the mobile phase is polar. This allows the separation and ultimately the quantification of highly polar, water soluble, low volatility compounds. This is an advantage over gas chromatography, which requires fairly low polarity, low boiling point samples.

In this instrument, the C18 column is packed with silica particles coated with a nonpolar 18-member alkyl chain. The nonpolar coating serves as the stationary phase. The mobile phase will be a combination of polar solvents, acetonitrile and water. When a sample is injected, the more polar compounds are not attracted to the stationary phase which causes them to elute through the column more quickly. The less polar components of the sample are retained on the column and elute at later times. Sometimes multiple components elute with very similar retention times. In such cases the polarity of the mobile phase can be altered to increase the separation. During the run, the mobile phase that elutes from the column is fed into a UV/Vis absorption instrument. An absorption measurement is taken periodically (approximately 10 measurements per second), and as species come off the column, a non-zero absorption shows up on the chromatogram. As each component in the sample elutes, a peak appears on the chromatogram which corresponds to the absorption of the excitation light (commonly 254 nm).
II.B. Solvents and Flow Rates Generally, the solvents used for these reversed-phase experiments are a combination of two of the following: water, methanol, acetonitrile, or tetrahydrofuran. For the experiments in this laboratory, different proportions of water and acetonitrile will be used. Generally, the flow rate is up to 1.0 mL/minute, and many of the test samples are run at 80% acetonitrile and 20% water. Increasing the amount of acetonitrile has the effect of decreasing the overall polarity of the mobile phase. II.C. Column Preparation To ensure that the column is prepared properly, it is necessary to monitor the baseline of the detector while at least 10 mL of the mobile phase flows through the column (for instance, if the total flow rate is 1.0 mL/minute, then the baseline monitoring should continue for at least 10 minutes). For example, if the mobile phase composition needs to be changed, then the necessary adjustments need to be made (such as changing the percentage of acetonitrile). II.D. Detector and Software The detector can simply be turned on, but it will require a few minutes to calibrate itself. An array of LEDs indicate the status of the machine.

Chromatographic Instrumentation: HPLC-3

CH 4200, Fall 2004 Prof. Greenlief

The software for this instrument is at ProgramsChromotagraphy32Karat. To start an experiment, double click the HPLC icon. It should take two to three minutes for the system to configure. Load the correct method for analysis. Lab 1 (Chem4200 Chromotographic Instrumentation) Lab 2 (Capsacin analysis)
II.E. Running a Sample. Load 32Karat software Start the Instrument "HLPC"

Open the Method Chem 4200 Chromotographic Instrumentation

The method gives the computer and instrument the default settings that are useful for this lab.

Access the Instrument Setup (or press F2)

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CH 4200, Fall 2004 Prof. Greenlief

Verify the Method time programming

Time programming instructs the instrument when to conduct specific actions. Here the lamp and flow start at 1mL/min for 10 minutes. At 10.0 minutes the data collection is complete. 10.10 minutes the lamp it switched off. And at 10.2 mintues it is confirmed that the pumps are running at 1ml/min.

Choose the solvent System

The Beckman 126 is configured such that 8 solvent systems can simultaneous be connected to the pumping module. This windows allows for selection of the solvent system used for this particular lab. Here is where the %composition can be changes of the solvent system substituents.

Configure the Detector analysis wavelengths

The Beckman 126 has a photodioide array detection system which allows for collection from 190-~500 nm. For analysis however the wavelengths of inteset need to be predetermined, and set in this window.

When done, click APPLY and manually close window.

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CH 4200, Fall 2004 Prof. Greenlief

Start a single run

This HPLC system is configured to work with an autosampler, which is located to the right of the pump module. Settings that need to be configured here include: Sample size 10-100ul, vial# on the autosampler tray and data file name.

The Chromatogram prints on screen as the instrument collects your data.

From this screen we are able to watch the experiment happen, and also after the experiment has finished running, from this window we conduct the analysis of the sample.

Chromatographic Instrumentation: HPLC-6

CH 4200, Fall 2004 Prof. Greenlief

1. 2. 3. 4. 5.

Set the threshold level (add to table) Set the minimum peak area (add to table) Define peaks for analysis Under the menu item Analysis, click Analyze View Custom report (or press F5)

Print a Chromatogram and table report for each run.

II.F. Test Samples and Benchmarks 5-component test mixture Before you begin the sample run you will want to perform a baseline monitor run to get an estimate of the noise in the detection system. Perform this operation, and make a note of the peak to peak noise level. You will need this to answer the questions at the end of the lab.

A solution containing all of the following compounds at the listed concentrations in 50:50 acetonitrile:water solvent has been previously prepared. Component Phenol Nitrobenzene Toluene o-Xylene p-Xylene Concentration (M) 9.73 x 10-4 M 5.0 x 10-5 M 3.8 x 10-3 M 2.0 x 10-3 M 1.0 x 10-3 M Concentration (mg/ml) 0.092 mg/ml 0.006 mg/ml 0.35 mg/ml 0.22 mg/ml 0.11 mg/ml

To illustrate the effect of mobile phase composition on the separation and length of the run, this mixture should be run at 50:50 and 80:20 acetonitrile:water. The 50:50 trial should be run for approximately 10 minutes, and then 80:20 trial should be run for approximately 8-10 minutes. Set the channels on the detector to monitor the 245 nm and 263 nm. In this case, acetonitrile is the less polar of the two components of the mobile phase. When a reversed phase (nonpolar) column is used, an increase in the percentage of acetonitrile results in shorter elution time, due to the fact that the less polar compounds that are solubilized on the stationary phase become more soluble in the mobile phase as it becomes less polar. The polarity index of the

Chromatographic Instrumentation: HPLC-7

CH 4200, Fall 2004 Prof. Greenlief

mobile characterizes the mobile phase polarity. The polarity index changes with composition, as illustrated in the following table. acetonitrile (%) 100 90 80 70 60 50 40 30 20 10 0 water (%) 0 10 20 30 40 50 60 70 80 90 100

Pcombined 5.80 6.24 6.68 7.12 7.56 8.00 8.44 8.88 9.32 9.76 10.20

The table above is based on the following equation:

Pcombined = acetonitirle Pacetonitrile + water Pwater


where Pcombined is the combined polarity index of the binary mixture, Pacetonitrile and Pwater are the polarity indices of the individual components of the mobile phase (5.80 and 10.20, respectively), and acetonitrile and water are the volume fractions mobile phase components. As the polarity index increases, the mobile phase becomes more polar, and the nonpolar compounds in the analyte mixture are more strongly retained. In each of the chromatograms, the peak heights and peak areas may seem somewhat deceiving. For example, even though nitrobenzene is the least concentrated of the species, it appears to be the largest peak in the chromatogram. This is due to the fact that nitrobenzene absorbs more strongly that the other components in the mixture, with nitrobenzene having an extinction coefficient of greater than 5000, and the other components having extinction coefficients of less than 500. Therefore, to make actual quantitative determinations, a series of standards of known concentrations for each component must be made in order to calibrate the measurement.

Chromatographic Instrumentation: HPLC-8

CH 4200, Fall 2004 Prof. Greenlief

Report Format

Make a table of retention time, peak height, peak width and peak area for each solvent composition at 245 and 263 nm, for a total of 4 tables.
Question 1: Explain why the retention time of the test mixture depends on the composition of the mobile phase. How can this be used to improve separation of compounds in liquid chromatography? Question 2: Calculate the number of theoretical plates for the first and last peaks of each chromatogram (i.e., the 50:50 mix and the 80:20 mix chromatograms). How do these compare with the GC plate heights? Question 3: Using the 80:20 chromatogram, calculate the sensitivity (in area per mg/ml) for each of the analytes from the 245 nm chromatogram. Why is this characteristic more meaningful for HPLC than for GC? (Hint: How is the sample volume on the column determined?) Estimate the limit of detection for each analyte in mg/ml. You should assume that the limit of detection is the point at which the peak height is equal to 3X the average noise level, and that the average noise level is one fifth of the peak-to-peak noise from the baseline monitor. Compute the minimum detectable peak, and compare this with the measured peak of each analyte of known concentration to estimate the limit of detection. How do these compare with the GC limits of detection?

Chromatographic Instrumentation: HPLC-9