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4.36: Chromatography II

  • Page ID
    123340
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    A laboratory is attempting to establish an HPLC assay for a drug (compound X). The assay employs an internal standard (compound I) for calculating the concentration of the drug. The initial HPLC conditions are as follows: Column type, C18; column temperature, ambient; flow rate, 2 mL/min (at 2000 psi); mobile phase, 30% methanol in water; and column effluent, monitored at 260 nm. The initial chromatogram for the standards, in which the initial peak is the internal standard and the second peak is compound X, is as follows:

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    The elution times for I and X are considered unacceptably longthat is, the k of the
    compounds are too large.

    QUESTIONS

    1. How can the elution time be reduced?
    2. The clinical chemist suggests that the medical technologist decrease the polarity index of the mobile phase to 70% of the current value in order to decrease the retention times and decrease the capacity factor (k). How should the technologist approach this problem?
    3. In order to obtain a mobile phase with a lower polarity index, the suggestion is made to prepare a mobile phase containing acetonitrile and chloroform. How can the technologist prepare the proper mobile phase with these new solvents?
    4. The new chromatogram produced using the mobile phase with the reduced polarity index calculated in question gives a ks that are now acceptable, but the resolution is now unacceptable. The clinical chemist now suggests replacing the chloroform by a solvent with the same polarity index but with a different chemical reactivity. Why was this suggestion made?
    5. The clinical chemist suggests preparing a new mobile phase consisting of a mixture of isopropanol and acetonitrile. What proportions of these two solvents should be used to maintain the same degree of polarity?

    Questions to Consider

    1. How would changes in the temperature of the column, polarity of the mobile phase, and flow rate affect the elution times from this column? How would changing these parameters affect the k' of the compounds?
    2. If the chromatographic conditions remain the same but the length of the bonded phase chain is decreased from 18 carbons (C18) to 8 carbons (C8), how would the elution time of I and X change?
    3. What is the polarity index of the currently constituted mobile phase?
    4. What is the minimum polarity index that can be obtained with methanol and water?
    5. Which combinations of chloroform and acetonitrile will give a polarity index approximately 70% that of the current mobile phase?
    6. What is the relationship of the solvent group to the chemical reactivity of the solvents? To the polarity index? What factor in chromatography theory affects separations on the basis of differences in chemical reactivity?
    7. Can solvents of the same polarity belong to different solvent groups?
    Answer
    1. In most cases, the most practical approach for reducing the elution time is to decrease, in the case of reversed-phase columns, the polarity of the mobile phase. Columns with different bonded phases may not be suitable for this analysis and may radically change the elution profile if used, and proper equipment for temperature equilibration of the column is rarely available in routine laboratories.
    2. Since the lowest polarity index possible with this reagent pair is greater than that desired (that is 5.8, or 70% of 8.28), a different set of reagents will need to be examined by the technologist.
    3. The final mobile phase will be 21.1% chloroform and 78.9% acetonitrite and will have a polarity index of 5.80. The initial approach the technologist will take is to change the polarity of the mobile phase using these new solvents according to the calculations described in questions 3 and 4. Decreasing the polarity by 70% is only an initial approximation. In actuality, a series of mobile phases with varying polarities, i.e. different proportions of strong and weak solvents, would be used to optimize retention time without totally decreasing resolution.
    4. The suggestion to maintain solvent polarity but change the chemical nature of the mobile phase is based on the usual chromatography practice of first optimizing the elution times and then changing the selectivity factor of the mobile phase in order to optimize resolution of solute peaks. Now that the retention time is acceptable, the technologist needs to improve resolution by changing the chemistry of the mobile phase.
    5. Since the polarity index of isopropanol and of chloroform are the same, and only their solvent group or chemical selectivity is different, the proportions of each in the mobile phase will remain the same, and the values calculated in question 4 can still be used: $$\begin{split} \text{isopropanol (or chlorofom)} &= 21.1 \% \\ \text{acetonitrile} &= 78.9 \% \end{split}$$

    Answers to Questions to Consider

    1. Increasing the column temperature will decrease the retention time; decreasing the polarity of the mobile phase will decrease the retention time in this reversed-phase system, and increasing the flow rate will decrease the retention time (pp. 115-117, 133). Only changes in temperature and mobile phase polarity can change the k. Increasing column temperature and increasing the polarity of the mobile phase will decrease the k.
    2. In reversed-phase HPLC, the chromatographic interaction is the partitioning of the salutes between the mobile phase and the bonded, apolar stationary phase. Shortening the length of the bonded hydrocarbon will increase the stationary phase’s polarity, reduce the interactions between solute and stationary phase, and decrease retention time (p. 117-118).
    3. Using the equation on p. 119, the polarity index can be calculated by summing the products of the volume fraction (\(\phi\)) and polarity index (P') of each of the constituents of the mobile phase (from Table 5-3, p. 120) (see also internet sites such as: http://home.planet.nl/~skok/techniques/hplc/eluotropic_series_extended.html#3, http://www.lc-ms.com/lcsolvent.htm , http://www.jtbaker.com/conversion/solventphydata.htm?#polar): $$\begin{split} P^{\prime}\; combined &= \phi_{methanol} P_{methanol}^{\prime} + \phi_{water} P_{water}^{\prime} \\ &= 0.3(6.6) + 0.7(9.0) \\ &= 8.28 \end{split}$$
    4. The least polar (strongest) mobile phase that can be obtained with methanol and water would employ only methanol. Therefore: $$P = 1.0 (6.6) = 6.6$$is the lowest polarity index possible.
    5. The final desired polarity index is 70% (8.28) or 5.80. Let x be the volume fraction of chloroform (polarity index, 4.3) and y be the volume fraction of acetonitrile (polarity index, 6.2). Thus: $$5.80 = x(4.3) + y(6.2)$$Since x + y = 1 and y = 1- x, $$\begin{split} 5.80 &= x(4.3) + (1-x)(6.2) \\ 5.80 &= 4.3x + 6.2 - 6.2x \\ -0.40 &= -1.9 x \\ 0.211 &= x = \text{volume fraction of chloroform} \\ and\; y &= 0.789 = \text{volume fraction of acetonitrile} \end{split}$$That is, in 100 mL of mobile phase, 21.1 will be chloroform and 78.9 mL will be acetonitrile.
    6. The polarity index of a solvent describes the overall interactive forces of that chemical. Basically, as the polarity of a chemical increases, its polarity index increases. The type of attractive force or chemical reactivity of each solvent is described by its solvent group (see pp 119-120). Thus all the solvents of group 9(IX) exhibit very strong hydrogen bonding and dipole properties while the solvents of group 0 have only temporary dipoles as their basis of interaction. There is no relationship between the polarity index of a solvent and its solvent group. The chromatographic factor that incorporates the concept of the solvent group is the alpha (\(\alpha\)) or selectivity factor (p.115).
    7. As mentioned in the previous answer, there is no relationship between polarity index and solvent group. Thus, both chloroform (polarity index, 4.3) and water (polarity index, 9.0) belong to the same solvent group [9(IX)].

    This page titled 4.36: Chromatography II is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by Lawrence Kaplan & Amadeo Pesce.

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