ESSENTIAL GUIDES TO METHOD DEVELOPMENT IN LIQUID …

1 4626 APPENDIX 2 / ESSENTIAL GUIDES TO METHOD DEVELOPMENT IN LIQUID CHROMATOGRAPHY. ESSENTIAL GUIDES TO METHOD DEVELOPMENT . IN LIQUID CHROMATOGRAPHY. J. W. Dolan and L. R. Snyder, LC Resources Inc., umns. For the present discussion, reversed-phase sep- CA, USA aration is assumed. The following section gives a brief Copyright ^ 2000 Academic Press description of the use of alternative HPLC modes for special samples. Once a mode is selected, the next step is to Rnd Introduction: Steps in METHOD conditions that will provide a separation of most DEVELOPMENT of the sample components. When this has been DEVELOPMENT of a METHOD for a high performance achieved, it is then possible to estimate the effort LIQUID chromatography (HPLC) separation can be that will be required to obtain an adequate separation a major undertaking.

2 Before the separation can be of all components. This Rrst step can be accomplished made, the sample must be in a suitable form to inject, using either gradient or isocratic elution. We favour and pretreatment steps are often required to remove an initial gradient run, because all peaks are likely to major interferences or materials that might shorten elute in a deRned time with reasonable separation of the column life. After conditions for adequate separ- both early and late peaks. Usually several isocratic ation are determined, some level of METHOD validation runs are required to achieve a similar result, and often is usually performed. Sample pretreatment and no isocratic conditions will provide an acceptable METHOD validation are beyond the scope of the pres- separation. From the initial gradient run it is possible ent discussion, which concentrates on achieving sep- to estimate whether isocratic elution is possible.

3 If aration. This article describes only the major steps this is the case, it is also possible to estimate condi- that are required for most samples. For additional tions that give reasonable separation of most sample information, the reader is urged to consult the refer- components. ence by Snyder et al. (see Further Reading) which As soon as this minimal separation is obtained, the covers HPLC METHOD DEVELOPMENT in detail. Addi- chromatogram should be examined for problems re- tional METHOD DEVELOPMENT information can be found lated to peak shape. Most obvious are peak tailing in the other monographs listed. problems. Although perfectly symmetrical peaks are preferred, many separations (usually for samples that contain basic compounds) will have one or more General Approach peaks that exhibit tailing. Most workers will accept There are different approaches to HPLC METHOD peaks with asymmetry factors, (United DEVELOPMENT , but we will follow the steps outlined States Pharmacopeia (USP) tailing factor, ).

4 In Table 1 and discussed below. For most samples, More severe tailing suggests the presence of un- this approach provides the highest probability of wanted sample interactions with the stationary phase. success with the minimum investment in time and The most common Rxes for tailing bands, in order of effort. decreasing usefulness, are: The Rrst step in HPLC METHOD DEVELOPMENT is to choose a chromatographic mode or METHOD type. The 1. the use of columns designed for the separation of most common modes are reversed-phase, normal- basic samples (based on very pure, type B silica);. phase, ion exchange and size exclusion. User surveys 2. adjustment of pH;. over the last 10 years consistently show that most 3. addition of triethylamine as a tailing suppressor;. separations are performed using reversed-phase col- 4.

5 Use of ion pairing;. 5. switching to a nonsilica ( polymeric) column. Table 1 General approach to HPLC METHOD DEVELOPMENT Symmetrical peaks that are too broad can also Select HPLC METHOD signal poor chromatographic behaviour; when Obtain minimal separation column plate numbers, N, for the sample are (60%. Check for and correct peak shape and width problems of the column manufacturer's test report. Broad Fine-tune primary variable peaks can result from the use of too strong a sample Change additional variables Adjust column conditions solvent, injection volumes that are too large, column overload or column problems. Usually it is advisable APPENDIX 2 / ESSENTIAL GUIDES TO METHOD DEVELOPMENT IN LIQUID CHROMATOGRAPHY 4627. Rrst to repeat the separation on a new column, to be sample resolution increases only slowly with decrease sure that the problem is caused by a bad column.)

6 In Sow rate or increase in column length, while run Reducing the injection volume to (25 !L, keeping time increases much faster. If resolution is greater the injected mass (10 !g, matching the injection than required, this means that an increase in Sow rate solvent with the mobile phase and increasing the and/or decrease in column length can be used for column temperature are some possible approaches to a signiRcant decrease in run time with acceptable loss sharpening broad peaks. in resolution. Smaller particle columns are typically Once acceptable peak shape is obtained, the next used in shorter lengths; these small particle columns step is to Rne-tune the primary variable: the percent- can provide shorter run times without loss in resolu- age of organic solvent in the mobile phase, %B, for tion or increase in column pressure.))

7 The column isocratic separations, or gradient time, tG, for gradi- pressure drop (or system pressure) increases with ent elution. In general, weaker (lower %B) isocratic higher Sow rates, longer columns and smaller par- mobile phases of shallower (larger tG) gradients will ticles. Since it is desirable to maintain a system pres- increase resolution at the expense of longer run times sure (200 atm, this places a further constraint on and broader peaks (with lower detection sensitivity). the latter column conditions. The best separation depends on the relative import- The simulated chromatograms of Figure 3 show ance of peak resolution, run time and detection sensi- the effect of changes in column conditions on the tivity, and will usually correspond to an intermediate aromatic sample of Figure 1. The lower run is the value of %B or tG.)

8 Same as the middle run of Figure 1, using a 250 mm, An example of the effect of isocratic solvent 5 !m particle column with a Sow rate of 2 mL min!", strength (%B) on retention and selectivity is seen in generating Rs" in 11 min with 100 bar back Figure 1 for the simulated separations of eight aro- pressure. By changing to a 150 mm, !m column matic compounds. It is seen that retention and band- at the same Sow rate, the run time is reduced to width increase inversely with %B. In general, Rs also 6 min. For many applications, the narrower peaks increases, but not for every peak pair } only seven out (and thus lower detection limits) and shorter run time of the eight peaks are visible at 70% and 50%B. Note will be worth the minimal loss in resolution and the relative forward movement of benzene from increase in pressure (Rs" , 120 bar back pres- 70%B, where it co-elutes with 2-nitrotoluene to sure).

9 If lower resolution is acceptable, a shorter col- 50%B, where it co-elutes with 2,6-dinitrotoluene; at umn (75 mm, !m) at a higher Sow rate intermediate solvent strengths it is resolved from (4 mL min!1) will reduce the run time to (2 min, as neighbouring peaks. shown in Figure 3C (Rs" , 120 bar back pres- Figure 2 shows the effect of gradient time (tG) sure). on retention and selectivity for simulated separations of a proprietary mixture of 11 herbicides. Retention and bandwidth increase with increasing tG. The over- Choice of HPLC Mode all resolution increases with longer gradients, but Reversed-phase HPLC will prove adequate for most note that peak 7, which elutes after peak 6 in the samples. Sample types requiring other chromato- 20 min gradient, moves ahead of peak 7 with longer graphic methods are summarized in Table 2.)

10 For gradient times. samples that fall in one of these categories, consult When satisfactory separation cannot be obtained the Further Reading section for detailed instructions. by adjustment of the primary variable (%B or tG), the usual problem is one of overlapping bands or selectiv- ity. In the latter case, other conditions (mobile phase, Choice of Starting Conditions column packing, temperature) can be varied. For A recommended set of starting conditions is sum- example, we recommend starting with acetonitrile as marized in Table 3. A C8 or C18 column is chosen, the B solvent. Changes in selectivity are often ob- with no particular preference for either phase. The served if methanol or tetrahydrofuran is used instead 150! mm column size packed with 5 !m particles of acetonitrile. Other variables worth examining are is capable of achieving most separations; with Sow column temperature, pH (for ionic samples), use of rates of 1}2 mL min!