Influence of the ionic strength of mobile phase on peak shape of antibiotics in RP-HPLC

 

mobile phase ionic strength

A new model that relates the retention of a weak acid in HPLC columns with the pH and ionic strength of the mobile phase is derived and tested for different benzoic acids in methanol−water mobile phases. The proposed model uses the pH value in the mobile phase instead of the pH value in water, takes into account the effect of the activity coefficients, and considers different holdup times Cited by: LC mobile-phase modifiers are used to improve peak shape and increase sample load tolerance. Trifluoroacetic acid (TFA) is a commonly used mobile-phase modifier, as it produces peptide separations that are far superior to other santacruzblog.ga by: This is accomplished by reducing the ionic strength of the buffer or decreasing the surface tension by adding ethyleneglycol or isopropanol to the mobile phase. It is also possible to displace the protein with detergents, though the effect is small and the problem of getting rid of them after chromatography has discouraged their use [ 65 ].



Read pages and and answer questions,and before coming to lab. The effects of solvent composition on retention of a series of anion standards is explored, mobile phase ionic strength.

Using retention information, a method to determine the amount of fluoride in toothpaste is developed. The HPLC measures fluoride by measurement of its conductivity after fluoride has been separated from other anions with the aid of anion exchange chromatography. Provided: Stock standards of fluoride, chloride, and nitrate,and stock solutions of carbonate mobile phase ionic strength bicarbonate have been prepared for you.

Solutions needed to prepare: Prepare mobile phase at the desired concentration recommended below. Make the solution described in the pre-lab. At the end of the experiment prepare an unknown solution of fluoride from toothpaste. See your mobile phase ionic strength for a method to extract the fluoride from toothpaste. Prepare the unknown on the day of use. Retention in HPLC is strongly dependent upon the composition of the mobile phase and somewhat less dependent upon the composition of the stationary phase.

In anion exchange chromatography, the column is filled with small diameter packing materials containing functional groups on the particle's surface that are easily ionized to be positively charged. When solvent molecules mobile phase is passed through the stationary phase anions from the solvent are weakly attracted to the positively charged stationary phase.

The solvent molecules may be displaced from the surface by other molecules which absorb more strongly. When analyte is absorbed to the stationary phase of the column the analyte is said to be retained.

Different analytes will exhibit different affinities for the stationary phase and will be retained for different amounts of time.

The mobile phase ionic strength of time each is retained on the column is called its retention time. The composition of the mobile phase will affect retention time of the analytes.

If a mobile phase is composed of relatively stronger absorbing anions, the analyte will not be attracted so strongly to the stationary phase, there will be mobile phase ionic strength net displacement of the solvent molecules by analyte and retention will be shorter. On the other hand, if the mobile phase is composed of relatively weaker absorbing anions, then analyte molecules will absorb to the stationary phase longer and retention times will be longer.

Ionic strength of the mobile phase is the most important variable for determining retention. Other variables that influence retention are pH, temperature, flow rate, organic modifies, mobile phase ionic strength, and, to some extent, stationary phase. What stationary phase are you using in this experiment? In this experiment you are going to vary the composition of the mobile phase ionic strength to see how that effects not only the retention of the analyte but also the retention of other anions present in the solution.

In the first part of the experiment you will be using a standard solution of F mobile phase ionic strengthCl -and NO 3. Determine the effect of changing the ionic strength of the mobile phase by making an eluent containing 21 mL of stock diluted to 1L.

The concentration of this is 3. Using the instructions for HPLC use available on this web site, set up a method which uses water and you eluent to give the optimum ionic strength. Inject the 5 anion standard. How well resolved were your peaks? Next, increase the retention by varying the mobile phase ionic strength. How will this be done? Next, mobile phase ionic strength, decrease the retention by varying the mobile phase composition.

Calculate resolution between fluoride and chloride, capacity factor, and number of theoretical plates for F - for each strength. Finally, try a gradient elution method which varies the ionic strength over the course of the chromatographic run to provide optimum resolution of all components in a minimum amount of time.

Now, try analyzing the concentration of fluoride in an unknown. To do this, mobile phase ionic strength, weigh approximately mg of toothpaste into a beaker.

After cooling quantitatively transfer into a mL volumetric flask and dilute to volume with ultrapure water. Develop an HPLC method to determine the amount of fluoride in toothpaste. Lab reports should be written with your lab partner. Each individual should submit a contribution form. The reports should be written up in the ACS format, suggested length, not including figures should be times number of weeks spent on the lab in pages.

Be sure to include references. Lab reports will be due one week after completion of the lab. In this experiment you studied the effect of ionic strength and, indirectly, pH on retention. In your report include the different mobile phase compositions you tried and their relative effect on resolution, retention, capacity factor, and number of theoretical plates.

Calculate resolution, retention, mobile phase ionic strength, capacity factor, and number of theoretical plates for each set of conditions tried. I did not have you use a different column stationary phase, mobile phase ionic strength. If you had used a different anion exchange column, how large of effect would that have had on retention? See the instructor for information on other stationary phases. Draw a schematic picture of the stationary phase you used.

In your report summarize your findings, answer any questions presented in the body of the experiment, and discuss any unusual occurrences. Also, include the method you used to quantitate fluoride in toothpaste and the amount you found present.

 

 

mobile phase ionic strength

 

A new model that relates the retention of a weak acid in HPLC columns with the pH and ionic strength of the mobile phase is derived and tested for different benzoic acids in methanol−water mobile phases. The proposed model uses the pH value in the mobile phase instead of the pH value in water, takes into account the effect of the activity coefficients, and considers different holdup times Cited by: LC mobile-phase modifiers are used to improve peak shape and increase sample load tolerance. Trifluoroacetic acid (TFA) is a commonly used mobile-phase modifier, as it produces peptide separations that are far superior to other santacruzblog.ga by: This is accomplished by reducing the ionic strength of the buffer or decreasing the surface tension by adding ethyleneglycol or isopropanol to the mobile phase. It is also possible to displace the protein with detergents, though the effect is small and the problem of getting rid of them after chromatography has discouraged their use [ 65 ].