Data Acquisition and Instrument Control Using the Turbochrom Chromatography Software. An Introduction to Аn introduction to high-performance liquid chromatography (HPLC): evaluating those experimental parameters that influence separations
5.14.1 Background and Summary of Method
Contemporary analytical instrumentation is said to be interfaced to computers. These developments commenced in the early to mid-1980s and took hold with Microsoft Windows® based software environments in the 1990s. The architecture for this technological advance can be illustrated as follows:
Interfaces can be either stand-alone or installed into the console of the PC. Instruments can be controlled and data acquired from a PC, or if a control is not available, only data acquisition is obtained. In our laboratory, both types of interfaces are used. With appropriate software, the control and data acquisition tasks are easily performed. If a means can be acquired to enable automatic sampling to be controlled as well, a totally automated system can be achieved! This was accomplished in our laboratory.
The HPLC within each student workstation is PC controlled, and the photodiode array detector (PDA) is interfaced to the same PC, thus enabling real-time data acquisition. Students are first asked to study the present architecture so as to gain an appreciation of contemporary HPLC- PDA-DS (data system) technology.
This experiment is designed to take students through an initial hands-on experience with the HPLC-PDA-DS from a first sample injection to a simple quantitative analysis. A quick method is first necessary for the software to recognize something. This is followed by optimizing the initial method, conducting a calibration, creating a customized report format, and evaluating the initial calibration verification standard (ICV).
Following completion of the initial experiment, the focus shifts to the separation of the test mixture or organic compounds using the HPLC instrument. The effect of solvent strength on k' and the effect of mobile-phase flow rate on Rs will be considered by retrieving previously developed Turbochrom® methods and making manual injections.
126.96.36.199 HPLC and TEQA
High-performance liquid chromatography (HPLC) followed GC in the early development of instrumental column chromatographic techniques that could be applied to TEQA. HPLC almost always complements and, depending on the analyte, sometimes duplicates GC. For example, polycyclic aromatic hydrocarbons (PAHs) can be separated and quantitated by both techniques; however, N-methyl carbamate pesticides can be determined only by HPLC as a result of the thermal instability in a hot GC injection port. Molecular structures for one example of each organic functional group are shown:
HPLC has become the dominant determinative technique for biochemists, pharmaceutical and medicinal chemists, yet has continued to take a secondary role with environmental chemists until technological advances led to the establishment, initially of HPLC-UV and HPLC-FL, followed by HPLC-PDA during the 1970s through to the 1990s. LC-MS first appeared followed LC-MS-MS technique in and around the early 2000s. Samples that contain the more polar and thermally labile analytes are much more amenable to analysis by HPLC rather than by GC. For example, a major contaminant in a lake in California went undetected until State Department of Health chemists identified a sulfonated anionic surfactant as the chief cause of the pollution. This pollutant was found using HPLC determinative techniques. HPLC encompasses a much broader range of applicability in terms of solute polarity and molecular weight range when compared with GC.
To illustrate how these different kinds of HPLC determinative techniques might aid the analyst in the environmental testing laboratory, consider the request from an engineering firm that wishes to evaluate the degree of phthalate ester contamination from leachate emanating from a hazardous waste site. Reversed-phase HPLC is an appropriate choice for the separation of lower- molecular-weight phthalate esters (e.g., dimethyl from diethyl from dibutyl). Attempts to elute higher MW and much more hydrophobic (lipophilic) phthalate esters e.g., dioctyl and bis (2- ethyl hexyl) under reversed-phase conditions were unsuccessful. The separation of these higher MW PAHs under normal-phase HPLC conditions was successful.
188.8.131.52 Flow-through Packed Columns
High-performance liquid chromatography requires that liquid be pumped across a packed bed within a tubular configuration. Snyder and Kirkland in their classic text on HPLC have used the Hagen-Poiseuille equation for laminar flow through tubes and Darcy’s law for fluid flow through packed beds and derived the following relationship:
where tQ is the retention time of an unretained solute (the time it takes after injection for an unretained solute to pass through the column and reach the detector), L is the length of the column, q is the viscosity of the mobile phase, AP is the pressure drop across the column, dp is the particle size of the stationary-phase packing, and /is an integer and is 1 for irregular porous, 2 for spherical porous, and 4 for pellicular packings.
The importance of stationary-phase particle size is reflected in the dependence of the void retention volume V0 = F • t0 where F is the mobile-phase flow rate in #cm3/min, (recall that 1 mL = 1 cm3) on the inverse square of d . Recall that the retention volume of a retained solute whose capacity factor is given by k' is
Hence, the smaller the dp, the larger is V0 and, consequently, VR. A smaller d also contributes in a significant manner to a larger N.
184.108.40.206 HPLC Also Refers to an Instrument That is a High-Pressure Liquid Chromatograph
It is quite useful to view the instrumentation for HPLC in terms of zones according to the following schematic:
Zone 1—Low-pressure zone prior to pump. This is a noncritical area served by Teflon tubing. A fritted filter is placed at the inlet to prevent particulates from entering the column.
Zone 2—High-pressure zone between pump and injector. This is a noncritical area served by standard stainless-steel (SS) tubing usually 1/16 inch in outer diameter (o.d.). A high-surface- area 0.5 pm filter can be placed here to prevent particulates from reaching the column.
Zone 3—High-pressure area surrounding injector and column. This is a critical area where the sample is introduced to the separation system. The volume must be well swept and minimized. Special fittings are 0.25 mm-inner diameter (i.d.) SS tubing.
Zone 4—Low-pressure area between column and detector. In this critical area, separation achieved in the column can be lost prior to detection. The volume must be well swept and minimized. Special fittings and 0.25 mm SS or plastic tubing are required. The critical zone extends to all detectors or fraction collectors in series or parallel connection.
Zone 5—Low-pressure area leading to waste collector. This noncritical area is served by Teflon tubing. Most labs fail to fit the waste vessel with a vent line to the hood or exhaust area.
High-performance liquid chromatograph (HPLC) instrument incorporating a UV absorption photodiode array detector (PDA) under reversed-phase conditions liquid chromatographic conditions.
220.127.116.11 Preparation of Chemical Reagents
Note: All reagents used in this analytical method contain hazardous chemicals. Wear appropriate eye protection, gloves, and protective attire. Use of concentrated acids and bases should be done in the fume hood.
- 18.104.22.168 Accessories to Be Used with the HPLC per Group
- 1 HPLC syringe. This syringe incorporates a blunt end; use of a beveled-end GC syringe would damage inner seals to the Rheodyne injector.
- 1 10 mL two-component mix at 1,000 ppm each. Prepare the mixture by dissolving 10 mg of ortho-phthalic acid (PhtA) and 10 mg of dimethyl ortho-phthalate (DMP) in about 5 mL of 50:50 (ACN: H,0) in a 50 mL beaker. After dissolution, transfer to a 10 mL volumetric flask and adjust to the final mark with the 50:50 solution. Molecular structure for PhtA and DMP are shown below:
Be sure to record your observations in your laboratory notebook.
22.214.171.124.1 Initial Observations of a Computer-controlled High-performance Liquid Chromatograph
Upon approaching the HPLC-PDA-DS, conduct the following:
- 1. Identify each of the five zones discussed above.
- 2. Locate the following hardware components:
a. The IEEE-488 cable to the LINK interface
b. The start/stop line from the Rheodyne injector to the LINK
c. The data acquisition line from the PDA to the LINK
d. The keying and master key.
126.96.36.199.2 Creating a QuickStart Method, Acquiring Data, Optimizing, Calibrating, and Conducting Analysis Using the QuickStart Method
Proceed with the Turbochrom 4 Tutorial and create a method using QuickStart. Inject an aliquot of a 100 ppm test mix reference standard. Optimize the method using the Graphic Editor. Develop the calibration and report format sections of your method. Establish a three-point calibration for DMP only between 10 and 100 ppm (always inject reference standards from low concentration to high concentration, never the reverse!) and prepare an ICV. Run the ICV in triplicate.
188.8.131.52.3 Effect of Solvent Strength on k'
A good practice when beginning to use a RP-HPLC instrument is to initially pass a mobile phase that contains 100% acetonitrile (ACN) so as to flush out of the reversed-phase column any nonpolar residue that might have been retained from previously running the instrument. Retrieve the Turbo method titled 100% ACN and download if not already set up. Download within “setup ” using the “method” approach.
Retrieve the method from Turbochrom or equivalent software titled 80% ACN and proceed to use Setup in the method mode to enable you to operate the HPLC with a mobile-phase composition of 80% ACN and 20% aqueous. The use of Setup is called downloading the method and sequence file so that data acquisition can begin. The aqueous mobile phase consists of
0.05% phosphoric acid (H,P04) dissolved in distilled deionized water (DDI). Carefully fill the 5 pL injection loop (the injector arm should be in the “load” position with the evaluation test mix with the HPLC syringe). Inject by moving the injector arm from the “load” position to the “inject” position. Observe the chromatogram that results and note the retention times of the components in the mixture. Give all members in the group the opportunity to make this initial injection.
Retrieve the method titled 60%ACN, download it. and then proceed to repeat the injection procedure discussed above. Obser>e the chromatogram that results and note retention times.
Retrieve the method titled 40%ACN, download it. and then proceed to repeat the injection discussed earlier. Observe the chromatogram that results and note retention times.
Retrieve the method titled 20%ACN, download it. and then proceed to repeat the injection procedure discussed earlier. Obser>e the chromatogram that results and note retention times.
184.108.40.206.4 Effect of Mobile-phase Flow Rate on Resolution
The mobile-phase flow rate will be varied and its influence on chromatographic resolution will be evaluated.
Retrieve the method titled FlowHi download it. and then proceed to use Setup as you did during the variation of solvent strength experiments. Allow sufficient equilibration time at this elevated mobile-phase flow rate. Notice what happens to the column back-pressure when a higher flow rate is in operation. Inject the test mix and observe the chromatogram that results.
Retrieve the method titled FlowLo download it. and then proceed to repeat the injection procedure discussed earlier. Obser>e the chromatogram that results.
5.14.3 For the Lab Notebook
The following empirical relationship has been developed for RP-HPLC. Refer to a theoretical discussion on HPLC or to a more specialized monograph.
where k' is the capacity factor for a retained peak, kw is the capacity factor (extrapolated) k' for pure water, Ф is the volume fraction of the organic solvent in the mobile phase, and S is a constant that is approximately proportional to solute molecular size or surface area.
Choose one component in the evaluation test mix and determine whether the above equation is consistent with your observations.
Address the following:
- 1. Among the three major parameters upon which resolution Rs depends, which of the three is influenced by changes in mobile-phase flow rate? Explain.
- 2. Mr. Everett Efficient believes that he can conserve resources by operating his HPLC using a mobile phase that consists only of a 0.01 M aqueous solution containing sodium dihydrogen phosphate (NaH,P04). Discuss what is seriously deficient in Mr. Efficient’s fundamental assumption.
- 3. Assume that you could change HPLC columns in this exercise and that you installed a column that contained 3 pm particle size silica. Assume that you used the same mobile- phase composition that you used for the reversed-phase separations that you observed.
Explain what you would expect to find if the reversed-phase test mix were injected into this HPLC configuration.
- 4. Explain why DMP is retained longer (i.e., has the higher k') than PhtA given the same mobile-phase composition.
- 5.14.4 Suggested Readings
To develop this experiment, the author consulted the following resources:
- • Guide to LC. Woburn, MA: Ranin Instruments Corporation.
- • Sawyer D., W. Heineman, J. Beebe. Chemistry Experiments for Instrumental Methods, New York: John Wiley & Sons, 1984, pp. 344-360.
- • Snyder L., J. Kirkland. Introduction to Modern Liquid Chromatography. 2nd ed. New York: John Wiley & Sons, 1979, pp. 36-37.
- • Ahuja S. Selectivity and Detectability in HPLC. Chemical Analysis Series of Monographs on Analytical Chemistry and Its Application, Vol. 104, New York: Wiley Interscience. 1989. p. 28.
- • Snyder L., J. Kirkland, J. Glajch. Practical HPLC Method Development. 2nd ed. New York: John Wiley & Sons, Inc. 1997.