The Retention Behavior of Reversed Phase hplc columns with 100% Aqueous Mobile Phase
Download 345.03 Kb. Pdf ko'rish
|
- Bu sahifa navigatsiya:
- Fig. 11.
|
Table 3. Effect of residual silanol groups a . Column Endcapping Hydrogen-bonding Capacity
b
Relative Amount of Residual Silanol Groups Final Retention Time Ratio c (%) Develosil ODS-A-5 No
1.42 Much
97.5 Develosil ODS-T-5 Single 0.53
Little 58.2
Develosil ODS-HG-5 Double
0.38 Very little 39.9 a. Conditions as shown in Fig. 3. b. Hydrogen-bonding capacity: separation factor of caffeine and phenol. c. The final retention time ratio is the final retention when the retention time decreased to the initial retention time.
Fig. 5. Effect of concentration of salt in the mobile phase. Column, 150 x 4.6 mm, 5 m dp C18, 10-nm pore size; mobile phase, ∆ = water, □ = 10 mM ammonium acetate (pH7.0), ○ = 100 mM ammonium acetate (pH7.0); column temperature, 40 ºC. Other conditions were the same as in Fig. 1.
6
1 mM ion-paring reagent showed drastic change in retention and the relative retention decreased to approximately 50%, it became stable after 10 minutes. On the other hand, a mobile phase with ion-paring reagent of normal concentration, which is 5 mM, the retention hardly changed and showed almost 100% of relative retention. Thus, it was confirmed that adding ion-paring reagent reduces retention changes and there became no change in retention at a concentration of 5 mM.
So far, the changes in retention time using water or phosphate buffers were discussed. Now, we will discuss the concentration of organic solvent. Fig. 7 shows different elution time of sodium nitrite as unretained compound using mobile phases of water added following solvents: methanol, acetonitrile, ethanol, dimethylformamide (DMF) and 2-propanol. The difference of elution time on the vertical axis shows times after the elution time was being deducted at 10% concentration from elution time of each concentration. The smaller values show the faster elution times. In other words, as described in the article discussing problems to use 100% aqueous mobile phase, the different elution times correspond to negative values of the mobile phase amount that was expelled from the packing material pores. The differences of elution times, that is, the expelled amount of mobile phase from pores of packing materials, greatly varied depending on the kinds of organic solvents. We confirmed that mobile phases were expelled from the packing material pores with less than 5% methanol, less than 2% acetonitrile and ethanol, and less than 1% 2-propanol. Although 1% of 2-propanol shows stable retention, the polarity of 2-propanol solution and 5% methanol solution is almost the same and therefore polar samples such as thymine would show almost the same retention time even using these mobile phases. Even though the retention of 2-propanol with low concentration to 1% is stable, the separation is almost as same as 5% methanol. Therefore, there is no merit to using 2-propanol. Put simply, the polarity of mobile phases with any organic solvents are the same as the concentration level of organic solvents when mobile phases are being expelled from packing material pores, and retention cannot be greater by changing the kinds of organic solvents under stable retention conditions.
The changes in retention time of 10-nm C18 and 10-nm C30 at different temperatures is shown in Fig.s 8 and 9. Conditions were the same as Fig. 2 except for stationary phases and temperatures. With 10-nm C18, the retention time decreased at 30 ºC and 40 ºC even while pumping. However, at 20 ºC, 10 ºC and 5 ºC, retention time did not change while pumping and it decreased only after pumping stopped. Furthermore, the lower the temperature was, the lower the decreasing retention time ratio. At the temperature lower than 10 ºC, the decreasing retention time ratio was less than 10% even after stopping the pump, and therefore it is possible to use this phase. Moreover, 10-nm C18, which was considered to be impossible to use, can realize separations with high reproducibility by setting
Fig. 6. Effect of concentration of the ion-pairing reagent in the mobile phase. Column, 150 mm x 4.6 mm, 5 μm dp C18, 10-nm pore size; mobile phase, ∆ = 10 mM sodium phosphate (pH7.0), □ = 10 mM sodium phosphate-1 mM sodium octanesulfonate (pH7.0), ○ = 10 mM sodium phosphate-5 mM sodium octanesulfonat (pH7.0). Other conditions were the same as in Fig. 1.
phase. Column, 150 mm x 4.6 mm, 5 μm dp C18, 10-nm pore size; mobile phase, ♦ = methanol/water, □ = acetnitrile/water, ▲ = ethanol/water, ○ = dimethylformamide (DMF), ∆ = 2-propanol; sample, sodium nitrite. Other conditions were the same as in Fig. 1.
7
column temperature below 30 ºC, switching to aqueous mobile phase and continuously pumping. This result matches the changes in retention time at different temperatures shown in Fig. 4. 10-nm C30 shows similar behavior to 10-nm C18, however the temperature range differs. 10-nm C30 did not change in retention time at 30 ºC even after pumping stopped. At 40 ºC, the retention time decreased by 5% after pumping stopped, and at 80 ºC, it decreases even while pumping and showed further decrease after pumping stopped. Compared to 10-nm C18, 10-nm C30 showed changes in retention at the higher temperature range of 40 ºC.
As aqueous mobile phase is expelled from the packing material pores, how retention would change was investigated when there is pressure around the packing material. The result is shown in Fig. 10. The condition is the same as Fig. 1. 10-nm C18 was used to measure the retention of 2-propanol at 40 ºC under aqueous mobile phase. Retention has completely decreased one hour after pumping stops. A tubing of ID 0.1mm was connected to the column outlet and back pressure was applied to the postcolumn. The back pressure was controlled by gradually extending the tubing from 0.2 m to 3 m without stopping pumping. The retention did not change when the back pressure was increased to 7.5 MPa after the retention decreased once. However, the retention suddenly increased between 10 MPa and 16 MPa, and gradually increased up to 30 MPa. The back pressure decreased from 30 MPa to 0 MPa without stopping the pump. According to the results in Fig. 2, 8 and 9, the retention changes over 10 hours during pumping. Therefore, the retention was measured after pumping more than 10 hours once the back pressure decreased. Up to 5 MPa, the retention did not change and decreased below 5 MPa. The condition of the aqueous mobile phase that is inside the packing material pores within the column is also shown in Fig. 10. It is considered that the retention increase and the increase of packing material with aqueous mobile phase permeating its pores occur at the same time. Aqueous mobile phase permeates inside of pores at a pressure of about 16 MPa. What needs to be looked at from this result is that greatly different curves were obtained when increasing and decreasing back pressure. This means that an extremely large hysteresis exists. Similarly, aqueous mobile phase sometimes permeates packing material pores at a back pressure of 5 MPa. This is determined by the condition before a back pressure of 5 MPa is applied. When the aqueous mobile phase permeates packing material pores, the mobile phase stays inside of the pores. On the other hand, when the aqueous mobile phase is not inside of the packing material pores, it does not permeate the pores even if a back pressure of 5 MPa was applied. Hysteresis such as this exists in nature, but the hysteresis effects on the aqueous mobile phase and C18 stationary phase is quite large. In order to measure pore size distribution of silica gel, an instrument, mercury intrusion porosimeter, is used. As mercury cannot be wet by any substance, a certain level of pressure is required to have it permeate pores. Pore size distribution can be determined by measuring the pressure and volume that mercury permeates
under 100% aqueous mobile phase conditions. Column dimensions, 150 mm x 4.6 mm; temperature, ○ = 5 ºC, □ = 10 º C, ■ = 20 º C, ▲ = 30 º C, ● = 40 º C. Other conditions were the same as in Fig. 2.
Fig. 9. Effect of temperature on C30 column (10-nm pore size) under 100% aqueous mobile phase conditions. Column dimensions, 150 mm x 4.6 mm; temperature, ○ = 30 ºC, □ = 40 ºC, ∆ = 80 ºC. Other conditions were the same as in Fig. 2.
8
pores. This relation is known as Washuburn’s [16] equation shown in formula (1).
Pr = -2γcosθ (1) The r is radius, γ is surface tension, θ is a contact angle and P is pressure.
In fact, the relationship between the C18 stationary phase and water is the same as silica gel and mercury mentioned above. Comparable curves can be obtained by shortening the column length, making the pressure difference close to zero between the column inlet and outlet, and differentiating the curve when back pressure is increasing under the same condition as shown in Fig. 10. When we measured the pore size distribution of silica material of 10-nm C18 stationary phase using this mercury intrusion porosimeter, the curve showing the relationship between the volume of mercury permeate pores and pressure was obtained, and this curve differed when pressure was increasing and decreasing. Thus, hysteresis exists. Both this hysteresis and the one shown in Fig. 10 were measured with the pores of the same silica gels and therefore there should not be a large difference on its pore shapes and distribution. However, it was confirmed that the hysteresis in Fig. 10 was much larger.
4. Wettability of the stationary phase and capillarity 4.1. Capillarity Capillarity [17] is a phenomenon that when a thin tube (capillary) is set in liquid, the liquid surface elevates higher than the tube or is depressed. Depending on the magnitude relationship between the cohesion of liquid molecular and adhesion between the liquid and capillary wall, the liquid surface elevates when liquid is wetting the tube (great adhesion) and is depressed when not wet. Capillarity can be expressed in the following formula having the h as the height differences in and out of the tube, r as the radius of the tube, ρ as liquid density, γ as the surface tension of liquid, θ as a contact angle and g as the acceleration of gravity.
h=2γcosθ/rρg (2) As octadecane of carbon number 18 cannot solve with methanol or water, the stearyl of C18 stationary phase is considered to be mixed in the methanol and aqueous mobile phase [18-20]. Therefore, the surface of C18 stationary phase within the packing material pores has a similar physical property as octadecane and does not get wet with water. As a result, a force to move out of the pores functions due to the capillarity. The aqueous mobile phase that is expelled from packing material pores by capillarity is considered to cause the retention decrease of C18 stationary phase when using the aqueous mobile phase. The previously mentioned Washburn equation fits the capillarity formula by transforming it and shows the same phenomenon. When changing the liquid explained in this capillarity exclusively to mercury, it applies for the Washburn equation. Majors [21] and his associates explain the necessary pressure for water to permeate the pores of
Fig. 10. Effects of the outlet pressure on the relative retention and water distribution in the column. Column, 150 mm x 4.6 mm, 5 μm d p C18, 10-nm pore size. Other conditions were the same as in Fig. 1.
9
dried reversed phase Solid Phase Extraction (SPE) packing material by using the Laplace-Young equation. This equation is the same as the Washburn equation when the radius r moves to the left, and shows the same as the capillarity formula. Originally, the Laplace–Young theory was derived from the observations of surface tension of substances, and as a result, their equation is the same as capillarity formula. Therefore, it is more appropriate to express the phenomenon that liquid being expelled from the pores in which the surface does not get wet as a capillary action is caused by capillarity rather than employing the theory of Laplace-Young. Based on the result shown in Fig. 10, the pressure that water permeates the pores of C18 stationary phase packing material with an average pore diameter 10 nm is approximately 16 MPa. Applying this value, the surface tension of 69.6 dyne cm-1 at 40°and 5.2 nm as the radius of pore diameter to the Washburn equation (1), the contact angle θ should be 126°. The contact angles can be calculated for C8 and C30 stationary phases under similar experiments. It is also possible to estimate contact angles from the data shown in Fig. 3. Based on the formula (1), which is the Washburn equation, supposing that the cosine function values of each pore diameter and contact angle are proportional when the relative retention time in Fig. 3 becomes 50%, the contact angles of the C8 and C30 stationary phases can be calculated based on the same C18 stationary phase. Pore diameters of C8, C18 and C30 stationary phases are 17.1 nm, 13.2 nm and 6.9 nm with 50% of relative retention time, and as the contact angle of C18 stationary phase is 126°, the same for C8 and C30 stationary phases would be 140° and 108°. Retention changes with reversed phase stationary phases under aqueous mobile phase are considered to be due to aqueous mobile phase that comes expelled from pores of packing materials because of capillarity, and parameters related to retention reproducibility can be explained based on capillarity. More aqueous mobile phase is expelled when pore diameters are small and less expelled when concentrations of salt and ion-pairing reagent are high with small liquid surface tension. It is also assumed that the contact angles change depending on alkyl chain lengths, ligand density of alkyl groups and the amount of residual silanol groups. As temperature increases, it is considered that liquid density, surface tension of liquid and contact angles change. Furthermore, it is thought that retentions recover by increasing the back pressure at the postcolumn outlet because higher back pressure is applied than the force that moves liquid out of the pores due to capillarity hence liquid moves back into the pores. In 1992, Montgomery and his associates [2] fixed the C18 stationary phase on a glass surface and measured the contact angle of water and the C18 stationary phase fixed on the glass surface. They found that the contact angle was 93° and reported that water does not wet the surface of the C18 stationary phase. However, in the discussion paragraph of the literature, they concluded that, as in previous cases, the retention decreases of the C18 stationary phase under 100% aqueous mobile phase occurs due to the ligand collapse of alkyl groups after all. It can be easily assumed that water is expelled from the pores due to capillarity when the contact angle is greater than 90°, however they did not reach this conclusion. That they have reached the same conclusion can be considered so as the curves expressing the relationship between pressures and retentions when the pressure around the packing material increases and decreases to show how aqueous mobile phase moves in and out of pores is greatly different as described in the previous section, thus there is a large hysteresis.
When using the mixed solvent of organic solvent and water as a mobile phase, it is considered that the organic solvent in the mobile phase is solvated [18, 19, 22-25] with stationary phases. Using C18 stationary phase after removing the solvated organic solvent, the retention reproducibility in aqueous mobile phase was investigated. Fig. 11 shows the result. Filling the inside of a column with chloroform that is packed with 10-nm C18 opening both column ends, we evaporated the chloroform in a chamber at
Fig. 11. Typical chromatograms of nitrite and nucleic acid bases. Column, 150 mm x 4.6 mm, 5 μm dp C18, 10-nm pore size; mobile phase, water; temperature, 40 ºC; detection, UV at 254 nm; flow rate, 1.0 mL/min; sample, 1 = sodium nitrite, 2 = cytosine, 3 = uracil, 4 = cytidine, 5 = uridine, 6 = thymine; A: C18 packing materials in a column were dried without plugs at 70 ºC for 10 hours after chloroform flowed in a C18 column. Then, water as a mobile phase flowed at 40 ºC at first and sample was injected into a column. B: Subsequently, 23 MPa of back pressure was added after outlet of a column without stopping flow. C: Flow was stopped for 1 hour, then flow was restarted. D: Successively, 23 MPa of back pressure was added after outlet of a column without stopping flow again.
10
70 ºC to dry the packing materials in the column. Then, we pumped aqueous mobile phase at 40 ºC into this column filled with dried C18 and injected the mixed samples of sodium nitrite and 5 kinds of nucleic acid bases. The chromatogram A shows the first injection of the sample after the baseline became stabilized and the 6 kinds of components eluted without being separated. From this condition, a tubing of ID 0.1 mm was connected to the postcolumn outlet without stopping the pump and applied the back pressure of 23 MPa to the column outlet. It is now assumed that aqueous mobile phase mixed with air came out of the column and residual air that existed in pores of the dried C18 packing materials was expelled. The chromatogram B shows the result of injection after the baseline stabilized. The retention time of thymine was approximately 8.5 minutes and all the six different of components were completely separated. Later, we stopped the pump for one hour and pumped again without applying pressure at the postcolumn outlet. The obtained chromatogram is shown as C, and the retention decreased by around 70%. Then 23 MPa of back pressure was applied again at the postcolumn outlet with continuously pumping. The first time, air came out of the column, however we could not confirm if there was air in the mobile phase that came out of the column the second time. Chromatogram D, which was obtained once the baseline became stabilized, was almost the same as chromatogram B. There was air in the pores of the C18 packing material with chromatogram A and approximately pressure of 6 MPa was applied at the column inlet side. However, with this pressure, aqueous mobile phase did not permeate the pores of the packing material and it is considered that it only came out between packing materials. Therefore, the interaction with more than 99% of C18 stationary phase in the pores did not occur and almost all the components were eluted without being retained. The elution time of sodium nitrite that is usually not retained is approximately 1.8 minutes. However, it was eluted at 1.1 minutes from chromatogram A proving the elution without permeating the pores of the packing material based on the elution time. As chromatogram B shows, by applying back pressure of 23 MPa, the aqueous mobile phase permeates pores, and the retention and elution time of sodium nitrite increased. Even if the aqueous mobile phase permeated the pores of the packing material, it was expelled from the pores once pumping stopped, and pressure around the packing material decreases to atmospheric pressure and the retention decreased as shown in chromatogram C. The retention increased again when 23 MPa back pressure was applied to the postcolumn outlet, but no air was expelled. This means that no air was in the pores of the packing material after the aqueous mobile phase moved out. Considering that the pressure that arises due to capillarity is more than a few MPa, it is easily estimated that the inside of the pores is close to a vacuum, which is -1 atmosphere. Therefore, as the water exits out of the pores, there is water vapor in the packing material pores and the water vapor pressure is believed to be the temperature.
The dispersion state of 10-nm C18 packing material to a mixed solvent of methanol and water is shown Fig. 12. Image C shows that 10-nm C18 packing material dispersed at 70% methanol and was wet. As the 70% methanol wets the surface of C18, the solvent permeates the pores due to capillarity. Image B shows that the packing material did not disperse by agitation only at 50% methanol but partially dispersed by applying ultrasonic vibration. As shown in Image A, the packing material did not disperse and wet at 30% methanol even when applying ultrasonic vibration. From this result, we confirmed that methanol wets the surface of C18 at more than 70% but not less than 50%.
We pumped methanol to a dried C18 column for 30 minutes to wet the C18 stationary phase and applied solutions of 10:90, 30:70, 50:50, 70:30 and 90:10 (v/v) methanol to water as mobile phases to investigate the elution time (t 0 ) of sodium nitrite and uracil that are believed to not be retained under the said condition. Sodium nitrite was used as a sample to measure each t 0
Download 345.03 Kb. Do'stlaringiz bilan baham: 
|