{"id":43691,"date":"2021-01-11T00:00:00","date_gmt":"2021-01-11T00:00:00","guid":{"rendered":"https:\/\/discover.restek.com\/uncategorized\/lc-method-translation-will-switching-column-dimensions-speed-up-sample-analyses\/"},"modified":"2026-03-18T13:41:07","modified_gmt":"2026-03-18T13:41:07","slug":"lc-method-translation-will-switching-column-dimensions-speed-up-sample-analyses","status":"publish","type":"post","link":"https:\/\/discover.restek.com\/es\/articles\/gnar3316\/lc-method-translation-will-switching-column-dimensions-speed-up-sample-analyses","title":{"rendered":"LC Method Translation: Will Switching Column Dimensions Speed Up Sample Analyses?"},"content":{"rendered":"\n

Introduction<\/h2>\n\n\n\n

Saving time, solvent, and sample through method translation is a very popular way to make improvements in liquid chromatography (LC) methods. For LC method translation, you need to keep the same particle type (e.g., Raptor) and stationary phase (e.g., Biphenyl), but it is possible that using a shorter, narrower column with a smaller particle size can offer big opportunities for speeding up your analysis while maintaining acceptable results. Those opportunities might mean higher sample throughput, more time for routine maintenance, or any number of other benefits that come from quicker methods using less mobile phase.<\/p>\n\n\n\n

But changing a column alone is not going to do the trick, especially in cases where separation and resolution of critical pairs is essential. You need to change the method conditions to match the new column so you don\u2019t lose critical resolutions, and that\u2019s where LC method translation calculators come into play.<\/p>\n\n\n\n

To achieve the goal of having very nearly the same separation in less time, you need to translate your column to one that has smaller dimensions, but that still maintains the same efficiency (typically expressed as a nearly equivalent total number of theoretical plates). However, it is not always practical or even possible to find a new column dimension that maintains the same number of plates as the original column (plus or minus 10%). That doesn\u2019t mean that a successful translation isn\u2019t possible; it just means that there will be some changes to your current separations. The new column and method might still help you meet your analytical goals in less time, so it is worth investing a little time with method translation software to explore your options.<\/p>\n\n\n\n

Let\u2019s walk through how you could use Restek\u2019s Pro EZ<\/em>LC method translator<\/a> to determine if switching a method to a new column will save time while still giving quality results.<\/p>\n\n\n\n

LC Method Translation Scenario<\/h2>\n\n\n\n

In this example, our lab has successfully developed a method using a 100 mm x 3.0 mm, 2.7 \u00b5m Raptor Biphenyl column. We also have a 50 mm x 2.1 mm, 2.7 \u00b5m column in house and\u2014knowing that moving to a column with smaller dimensions may decrease the analysis time\u2014we want to know whether the current method on the larger column could be successfully translated to the shorter, narrower column.<\/p>\n\n\n\n

In this particular case, the smaller column represents a significant loss in total theoretical plates. That means we should not expect the exact same separation in less time with a properly translated method, so we need to be careful that we do not lose critical resolutions as a result of LC method translation. But, even though we know we won\u2019t get the same separation, will we still get acceptable chromatographic results?<\/p>\n\n\n\n

Calculating new method conditions and predicting the new results is exactly what a method translation calculator is designed to do. So, let\u2019s see how it works.<\/p>\n\n\n\n

Method Translation Example<\/h2>\n\n\n\n

We want to translate the analysis of certain drugs of abuse to a smaller dimension column to see if we can further decrease our analysis time without sacrificing performance even though we\u2019re happy with the chromatographic performance provided by the original, longer column. Of the 15 drugs of abuse, there are five pairs of isobars that require chromatographic separation because the MS\/MS would not be able to differentiate them. The original method does that, so we are particularly interested in whether the translated method will as well. Figure 1 below shows the results of the original method on the 100 mm x 3.0 mm column with 2.7 \u00b5m particles.<\/p>\n\n\n

Figure 1:<\/strong>\u00a0Chromatographic results using the original column dimensions and method.<\/div>
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\"Original<\/div>

LC_CF0756<\/p>

Peaks<\/h4>\n
<\/th>Peaks<\/th>tR<\/sub> (min)<\/th>Precursor Ion<\/th>Product Ion<\/th><\/tr><\/thead>\n
1.<\/td>Morphine<\/a><\/td>1.11<\/td>286.2<\/td>152.1<\/td><\/tr>\n
2.<\/td>Hydromorphone<\/a><\/td>1.43<\/td>286.2<\/td>184.9<\/td><\/tr>\n
3.<\/td>Norcodeine<\/a><\/td>1.86<\/td>286.1<\/td>151.9<\/td><\/tr>\n
4.<\/td>Norhydrocodone<\/a><\/td>2.49<\/td>286.1<\/td>199.0<\/td><\/tr>\n
5.<\/td>6\u03b2-Naltrexol<\/a><\/td>3.32<\/td>344.3<\/td>326.1<\/td><\/tr>\n
6.<\/td>Tramadol<\/a><\/td>4.42<\/td>264.2<\/td>58.0<\/td><\/tr>\n
7.<\/td>Normeperidine<\/a><\/td>4.82<\/td>234.1<\/td>160.2<\/td><\/tr>\n<\/tbody><\/table>\n\n
<\/th>Peaks<\/th>tR<\/sub> (min)<\/th>Precursor Ion<\/th>Product Ion<\/th><\/tr><\/thead>
8.<\/td>Mirtazapine<\/a><\/td>5.50<\/td>266.1<\/td>195.1<\/td><\/tr>\n
9.<\/td>Clozapine<\/a><\/td>5.87<\/td>328.2<\/td>271.1<\/td><\/tr>\n
10.<\/td>Pentazocine<\/a><\/td>5.95<\/td>286.2<\/td>218.1<\/td><\/tr>\n
11.<\/td>7-Aminoflunitrazepam<\/a><\/td>6.50<\/td>284.1<\/td>135.0<\/td><\/tr>\n
12.<\/td>Fluoxetine<\/a><\/td>7.53<\/td>310.1<\/td>148.0<\/td><\/tr>\n
13.<\/td>Loxapine<\/a><\/td>7.86<\/td>328.1<\/td>271.1<\/td><\/tr>\n
14.<\/td>EMDP<\/a><\/td>8.28<\/td>264.2<\/td>235.2<\/td><\/tr>\n
15.<\/td>Thioridazine<\/a><\/td>8.87<\/td>371.2<\/td>126.1<\/td><\/tr>\n<\/tbody><\/table><\/div>

Conditions<\/h4>
Column<\/th>Raptor Biphenyl (cat.# 9309A1E<\/a>)<\/td><\/tr>
Dimensions:<\/th>100 mm x 3.0 mm ID<\/td><\/tr>
Particle Size:<\/th>2.7 \u00b5m<\/td><\/tr>
Pore Size:<\/th>90 \u00c5<\/td><\/tr>
<\/td>
Temp.:<\/th>40 \u00b0C<\/td><\/tr>
Standard\/Sample<\/th><\/td><\/tr>
Diluent:<\/th>Water<\/td><\/tr>
Conc.:<\/th>0.5-10 \u00b5g\/mL<\/td><\/tr><\/td><\/tr>
Inj. Vol.:<\/th>5 \u00b5L <\/td><\/tr>
Mobile Phase<\/th><\/td><\/tr>
A:<\/th>Water, 0.1% formic acid <\/td><\/tr>
B:<\/th>Methanol, 0.1% formic acid <\/td><\/tr>
<\/td>
Time (min)<\/th>Flow (mL\/min)<\/th>%A<\/th>%B<\/th><\/tr><\/thead>
0.00<\/td>1.0<\/td>85<\/td>15<\/td><\/tr>
6.50<\/td>1.0<\/td>50<\/td>50<\/td><\/tr>
10.00<\/td>1.0<\/td>0<\/td>100<\/td><\/tr><\/tbody><\/table><\/td><\/tr><\/table>
Detector<\/th>MS\/MS<\/td><\/tr>
Ion Source:<\/th>Electrospray <\/td><\/tr>
Ion Mode:<\/th>ESI+ <\/td><\/tr>
Mode:<\/th>MRM <\/td><\/tr>
Instrument<\/th>UHPLC<\/td><\/tr>
Sample Preparation<\/th>A standard mix with 15 drugs of abuse was prepared in concentrations ranging from 500-10,000 ng\/mL in water. The solution was vortexed at 3000 rpm for 10 seconds to mix, and the supernatant was injected for LC-MS\/MS analysis.
<\/td><\/tr><\/table><\/div><\/div><\/div>
<\/path><\/g><\/path><\/g><\/svg>Download PDF<\/a><\/div><\/div><\/div>\n<\/div><\/div><\/div>\n\n\n

<\/p>\n\n\n\n

Our next step is to provide the LC method translation calculator with information about our current column and method so it can provide the best translated method conditions for the new column dimensions. Note that when using this calculator, you must be transferring between columns of the same particle type, for example FPP to FPP (fully porous particle) or SPP to SPP (superficially porous particle). Figure 2 illustrates entering the original column dimensions, injection volume, dwell volume, \u201cextra-column volume effect\u201d value, and the mobile phase conditions, which is a gradient analysis in this case.<\/p>\n\n\n\n

Take special note of the default dwell volume and extra-column volume effect values because, while they may not be your instrument\u2019s values, they are particularly important because the translator will take them into account when determining values like critical pair resolutions and compound retention times under the translated run conditions. If you don\u2019t know these values for your particular instruments, look up this information in your instrument documentation, contact the instrument manufacturer, or research ways to determine these values empirically. You can find out more about these values in the translator\u2019s glossary, which you can access by clicking the \u201c?\u201d button in the upper right corner of the translator.<\/p>\n\n\n

Figure 2:<\/strong>\u00a0The first step of using an LC method translator: entering your current column, instrument, and method details.<\/div>
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