a strategy to develop fast rp-hplc methods using monolithic silica columns

J. Sep. Sci. 2007, 30, 1993 –2001 S. El Deeb et al. 1993

Sami El DeebLutz PreuHermann W�tzig

Institute of PharmaceuticalChemistry, TU Braunschweig,Braunschweig, Germany

Original Paper

A strategy to develop fast RP-HPLC methods usingmonolithic silica columns

Since the appearance of monolithic silica, much work has been done describing theproperties of monolithic silica columns. Meanwhile the transferability of analyticalmethods from conventional to monolithic silica columns has been intensively inves-tigated [1–5]. RP HPLC method development strategies for conventional columnsshould be updated or scaled to meet the higher performing monolithic columntechnology. Because of the high permeability of monolithic silica columns it shouldbe possible to decrease the time for method development by applying high isocraticflow rates. Here we suggest a clear strategy for method development using mono-lithic columns. The strategy will be applicable for various sample compositions, e. g.,acidic, basic, or neutral. The applicability of monolithic columns for especially com-plex separations of basic mixtures without the need of using a highly basic mobilephase that harms the column will be pointed out in this work. This work willdescribe in detail the actual method development process. For better understandingof our strategy, the influence of flow rate, column length, mobile phase composi-tion, pH, and temperature will be discussed. Details about the application of a flowprogram will be mentioned.

Keywords: Fast HPLC / Flow programming / Method development strategy / Monolithic columns /

Received: March 2, 2007; revised: March 30, 2007; accepted: April 23, 2007

DOI 10.1002/jssc.200700092

1 Introduction

Standard LC method development for the separation oftest mixtures of unknown composition suggested theuse of a high percentage organic modifier for the initialruns, a flow rate of up to 2 mL/min, a suitable columnwith a suitable temperature in the range between roomtemperature and about 458C, and a sample size of lessthan 50 lL. Both isocratic and gradient methods havebeen used for standard HPLC method development. Ini-tial gradient elution is often used to determine the bestisocratic conditions. Variation of % organic modifier isthe corner stone for the optimization of band spacingand resolution using standard particle-packed columns[6].

More options are offered for method developmentusing monolithic columns, because of the highly porousnature of monolithic silica columns and the ability toconnect columns together. Variation of the flow rate,connecting columns together, and the use of flow pro-

gramming provide additional variation parameters forthe optimization of band spacing and resolution duringmethod development other than changing the percent-age of the organic modifier and the column type. Fur-thermore, the use of high flow rate saves time for condi-tioning, washing, and equilibrating the column duringmethod development. All these advantages should aid inthe reduction of the total time to develop a method usingmonolithic silica compared with conventional particlepacked columns.

Investigations on method development require realseparation problems. If there is a developed or existingstandard LC method, the researcher is automaticallyinfluenced and will come to a biased decision. In order tosolve a real separation problem, we have thought ofusing recently registered drugs for which no previousstudy has been reported. However, recently registereddrugs and their impurities were not made available bythe pharmaceutical companies. Thus, we composed mix-tures to which no methods have been developed before.A mixture consisting of nicotinic acid, resorcin, phenol,salicylic acid, benzoic acid, 4-hydroxy-benzoic acid, and2-naphthol was chosen as an acidic mixture. Two basicmixtures were also made. The first consisted of aniline,N-methylaniline, N-ethylaniline, 4-ethylaniline, di-

Correspondence: Professor Dr. Hermann W�tzig, Institute ofPharmaceutical Chemistry, Technical University Braunschweig,Beethovenstrasse 55, D-38106 Braunschweig, GermanyE-mail: [email protected]: +49-531/391-2799

i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

1994 S. El Deeb et al. J. Sep. Sci. 2007, 30, 1993 – 2001

methylaniline, and N,N-diethylaniline. The second basicmixture was an alkaloid mixture of codeine phosphate,ephedrine HCl, theophiline ethylenediamine, atropinesulfate, yohimbine HCl, butylscopolamine bromide, andpapaverine HCl.

2 Experimental

2.1 Chemicals and reagents

The following substances nicotinic acid, resorcin, phe-nol, salicylic acid, benzoic acid, 4-(OH) benzoic acid and2-naphthol, aniline, N-methylaniline, N-ethylaniline, 4-ethylaniline, dimethylaniline and N,N-diethylaniline,codeine phosphate, ephedrine HCL, theophiline ethyl-enediamine, atropine sulfate, yohimbine HCl, papaver-ine HCl, and butylscopolamine bromide were purchasedfrom different pharmaceutical companies and were ofanalytical grade. ACN of HPLC grade was purchased fromAcros Organics (Gelnhausen, Germany). Water for HPLC/LiChrosolv was purchased from Merck (Darmstadt, Ger-many). Potassium dihydrogen phosphate, sodium dihy-drogen phosphate dihydrate, disodium hydrogen phos-phate, and phosphoric acid (85%) were purchased fromRiedel-de-Ha�n (Seelze, Germany),

2.2 Instrumentation

Analyses were performed on a Merck-Hitachi HPLC sys-tem consisting of a solvent pump (model L 6200 A), anAutosampler (AS 2000A), a UV-Vis detector (L-4250), andan Interface (D-6000). The data were collected and ana-lyzed using the D7000 HSM software (Merck).

2.3 Chromatographic conditions

Separations were performed on Chromolith Perform-ance RP-18e columns (10064.6 mm, Merck) with theability to connect columns together using a specialmonolithic column coupler. Different isocratic flowrates from 1 to 9 mL/min were used throughout themethod development. Moreover, different flow rate gra-

dients (flow-rate programming) which involve a stepwiseincrease in the flow rate using one pump according to adefined program were used depending on the resolutionbetween peaks.

Mobile phases were prepared by mixing ACN HPLCgrade and/or methanol HPLC grade in the range between80 and 5% with phosphate and citrate buffers at differentpH values that cover the column pH stability range(pH 2–7.5) Table 1 shows the preparation of different pHbuffers based on a previous work [7]. Temperature in therange from room temperature up to 458C has been triedfor method development.

The column was equilibrated by running at the flowrate of 9 mL/min for 3 min, and then by returning to theflow rate of 5 mL/min.

Samples of less than 25 lL and less than 10 lg (of eachanalyte) were injected.

3 Results

The steps for RP-HPLC method development using mono-lithic silica columns have been summarized in Chart 1.The strategy depends on using a relatively high flow ratefor method development to reduce time and effort.

Examples outlined in this section show how mono-lithic columns can provide fast RP-HPLC method develop-ments which would not be possible with conventionalcolumns. The first example shows the separation of sevenacidic compounds using monolithic silica columns. Aninitial flow rate of 5 mL/min was used with a mobilephase consisting of ACN/phosphate buffer (80:20, pH 2).The chromatogram obtained under this conditionshowed only one peak for the whole mixture indicatinga coelution of the compounds altogether. Successiveruns were tried with 60, 40, 20, 10, and 5% ACN in themobile phase. The best separation was obtained by 10%ACN but only five peaks appeared for the seven com-pounds because nicotinic acid, resorcin, and phenolwere coeluted together (Fig. 1a). No further improvementwas obtained by decreasing ACN to 5%. On the otherhand, no better separation was obtained by increasingthe pH to 3, 4, or 5 using ACN as an organic modifier.


Table 1. Buffer preparations

Standard solutions used for thepreparation of 2 L buffer solution

pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 pH 7.4 pH 8

0.1 M phosphoric acid 6.86 mL/L 282.5 55.0 – – – – – –0.1 M acetic acid 5.8 mL/L – – 410.0 148.0 – – – –0.1 M sodium acetate solution8.2 g/L

– – 90.0 352.0 – – – –

0.1 M sodium dihydrogenphos-phate dihydrate solution 15.6 g/L

217.5 445.0 – – 438.5 195.0 95.0 26.5

0.1 M disodium hydrogenphos-phate dihydrate solution 17.8 g/L

– – – – 61.5 305.0 405.0 473.5

J. Sep. Sci. 2007, 30, 1993 –2001 Liquid Chromatography 1995

After replacing the best obtained ratio of ACN (10%) bymethanol (methanol/phosphate pH 2, 10:90), also onlysix peaks appeared because the peaks for salicylic andbenzoic acids overlapped (Fig. 1b). The best separationwas obtained using pH 3 and 10% methanol in themobile phase (Fig. 1c). Further reduction in the run timewas achieved using an appropriate flow program (Table2) as shown in Fig. 1d. Figure 2 shows the separation of abasic mixture of aniline and five derivatives. As a startingcondition, a mobile phase of methanol/buffer (80:20,pH 2) was used. Best resolution was obtained after reduc-ing methanol to 45% and increasing the pH to 5. Underthese conditions, the six compounds were separatedwithin 4.5 min using the flow program mentioned inTable 3 as shown in the chromatogram of Fig. 2. Figure3a shows the separation of a challenging basic alkaloidmixture. This chromatogram was the best one obtainedafter trying different pH values from pH 2 to 8 in the

mobile phase. However, peaks 1, 2, and 3 are still unre-solved. Resolution of this basic mixture seems to requirea high basic pH value in the mobile phase which inter-feres with the column stability. However, as shown in(Fig. 3b), a satisfactory separation of peaks 1, 2, and 3 hasbeen achieved by increasing the separation efficiencythrough connecting the two columns together. Althoughthis has doubled the run time in the first place, we havesubsequently reduced the run time again by applying


Chart 1. Strategy for method development using monolithic silica columns.

Table 2. Flow program used for the separation of acidic mix-ture in Fig. 1d

Time (min) Flow rate (mL/min)

0.0 51.0 61.1 98 9


the flow program mentioned in Table 4, as shown in thechromatogram of Fig. 3c.

4 Discussion

Nowadays, there is a competition between two methodsfor fast LC analysis, namely, HPLC with monolithicphases and small particle phases used in ultra Perform-

ance LC (UPLCTM). With both phase types, a substantiallyhigher column efficiency, analysis speed, and sensitivitycan be achieved. With UPLC this is achieved particularlyby the use of small particles in the stationary phases.However, the use of small sub-2 lm particles requires theuse of ultra high pressure (approximately 1400 bar =20 000 psi) demanding a special high quality equipment


a b

c d

Figure 1. (a) Separation of seven acidic compounds. Column: Chromolith Performance; eluent: ACN/25 mM phosphate bufferpH 2 (10:90). Flow rate: 5 mL/min and detection wavelength 273 nm. Nicotinic acid (1), resorcin (2), phenol (3), salicylic acid (4),benzoic acid (5), 4-(OH) benzoic acid (6), and 2-naphthol (7). (b) Separation of seven acidic compounds. Column: ChromolithPerformance; eluent: methanol/25 mM phosphate buffer pH 2 (10:90). Peak names as in Fig. 1a. (c) Separation of seven acidiccompounds. Column: Chromolith Performance; eluent: methanol/25 mM phosphate buffer pH 3 (10:90). Flow rate: 5 mL/min anddetection wavelength 273 nm. Peak names as in Fig. 1a. (d) Separation of seven acidic compounds. Column: Chromolith Per-formance; eluent: methanol/25 mM phosphate buffer pH 3 (10:90) using the flow program shown in the Table 2. Peak names asin Fig. 1a.

Table 3. Flow program used for the separation of aniline andits derivatives in Fig. 2


0.0 3.02.1 3.02.2 9.05.0 9.0

Table 4. The flow program used for the separation of thealkaloid mixture in Fig. 3c


0.0 3.03.5 3.05.0 5.05.1 9.018 9.0


to cope with the high pressures. While for UPLC manyphases are already commercially available, the selectionof available monolithic phases is still limited at presentto standard materials such as RP-18 and RP-8. However,these phases do have a substantially higher permeabilityto build a much lower backpressure in comparison tothe small particle phases used in UPLC. Therefore mono-lithic phases can be used with standard HPLC instru-ments [8].

In a comparison between HPLC and UPLC carried outby Waters Company, the solvent consumption has beenreported as a disadvantage of using monolithic columns[9]. According to our work, the increase in solvent con-sumption of monolithic columns at high flow rates wastotally compensated by the decrease in the chromato-graphic run time. Of course this is more than the solventused in UPLC where low flow rates are sufficient for sep-aration because of the small column diameter.

The parameters that control separation in monolithicsilica columns are the same for conventional silica col-umns and for RP HPLC separation in general. However,the effect of certain parameters on the separation maydiffer. For example, a parameter such as flow rate plays amore important role in monolithic silica columns thanin conventional particle packed-columns. On the otherhand, parameters such as organic modifier and pH of themobile phase play nearly the same role in the two col-umn types.

The stationary phase which is an important parameterto affect separation has been fixed here to monolithic RP-18e column. However, there is a possibility to change thecolumn length by connecting several columns togetherusing a column coupler. Due to high column porosity,the added column backpressure and the prolonged anal-ysis time can be compensated by flow programming.

Increase in the column length will be particularly impor-tant for complex basic mixtures (compare Figs. 3a and b)at which the resolution between different compoundsnecessitates the use of high pH mobile phase which isnot suitable with silica columns. The use of flow rate gra-dients (flow rate programming) in LC separations hasbeen previously reported, especially in microcolumnHPLC [10–18].

Flow rate is an important separation parameter thatmakes a great difference between method developmenton conventional and monolithic silica columns. Thehigh permeability offered by the high porosity of the col-umn allows the use of high flow rates without the devel-opment of a significant backpressure. A standard LCmethod can be converted to a fast LC method on mono-lithic columns, only by increasing the flow rate. In mono-lithic columns, the increase in flow rate does not lead tosubstantial losses in resolution because of the low masstransfer resistance compared to conventional particle-packed columns [19]. Flat curves for plate height versuslinear velocity were obtained while analyzing drugs withmonolithic silica column [20]. Silica monoliths also havea high hydrodynamic permeability and favorable separa-tion impedance. This provides a time-saving methodwith minimal loss of resolution. This could be doneeither by applying a high isocratic flow rate or using flowrate programming.

When some compounds of the analyzed mixture haveclose retention time values, while others have not, loss ofresolution between the closely related peaks limits theability for further reduction in analysis time. In suchcases, further reduction in chromatographic run timecould be achieved by the application of gradient elutionor flow rate programming. Flow programming isdependent on the use of a relatively low flow rate at that


Figure 2. Separation of six basiccompounds, aniline, and five deriv-atives. Column: Chromolith Per-formance RP-18e; eluent: 25 mMphosphate buffer pH 5/methanol(55:45); detection wavelength214 nm, using the flow programshown in Table 3. (a) Aniline, (b)N-methylaniline, (c) N-ethylaniline,(d) 4-ethylaniline, (e) dimethylani-line, (f) N,N-diethylaniline.


chromatographic part where the resolution is criticaland increasing the flow rate speed at periods duringwhich no peak appears or peaks are far away from eachother, as shown in Figs. 1e, 2, and 3c. In gradient elution,the solvent polarity (composition) is continuously variedor stepped. A system for mixing and degassing themobile phase must be used. Furthermore, the HPLC gra-dient mixers must provide a very precise control of thesolvent composition to maintain a reproducible gradientprofile. The use of flow rate gradient (flow rate program-ming) in HPLC separations involves a stepwise increasein the flow rate using one pump according to a definedflow program. As an important advantage of flow rateprogram over gradient elution of mobile phase, equili-

bration of the system is not required after each separa-tion. This is important to achieve fast analysis of a seriesof samples. Flow rate programming is more suitable formonolithic than conventional particle-packed columnsdue to higher permeability and lower backpressure.Instrumentation failure due to a high column backpres-sure usually occurs when flow programming is appliedon conventional particle-packed columns [21].

As with conventional columns, decrease in the percent-age of organic modifier will decrease the elution strength,increase the retention time, and improve resolution. ACNand methanol will be selected as the first choice organicmodifiers. THF, which is a possible solvent in RP chroma-tography, will be avoided due to its incompatibility with



the long term use with PEEK tubing of the monolithic col-umn in addition to its well-known disadvantages likehigh absorbance and reactivity with oxygen.

Too much water in the mobile phase can collapse thebonded phase, even though we have obtained a reliableseparation for pilocarpine analysis on monolithic col-umn with as little as 2% methanol in the mobile phase[20].

As for conventional silica columns, the pH stability formonolithic columns ranges from pH 2 to 7.5. For acidiccompounds, one will probably succeed in achieving afull resolution between the individual compounds usingan acidic mobile phase. They will be unionized at pH 2and thus better retained with the possibility to obtainthe ionizable form for some component by raising thepH in the acidic range to improve the selectivity. At pHvalue of more than l1.5 of the pKa, the compound will beeither almost completely ionized or unionized [22].

For basic compounds, it is also better to achieve separa-tion in the acidic or mild pH ranges because of two rea-sons; first because the silica backbone is soluble at a highpH value. Another reason is the decrease in secondaryinteraction between basic compounds and ionized sila-nol groups of the silica column which leads to an exten-sive peak tailing. This effect is minimized at acidic pH atwhich silanols are nonionized.

However, as some basic compounds have high pKa val-ues (as some alkaloids), it could be difficult to obtain fullresolution between the individual components withoutusing a high pH mobile phase, which in turn is problem-atic with silica-based RP-18 columns for the above-dis-cussed reasons. In this case, it might be an advantageousstrategy to step back to mild pH conditions and toimprove only a partial separation of peaks by column cou-pling. The increase in column length will gain a better res-olution; the augmented run time can then be reduced byapplying a proper flow rate program. The increased runtime due to increased column length could then bereduced by applying a proper flow rate program.

Increase in temperature in RP HPLC leads to a decreasein peak broadening and retention time [23–25]. How-ever, this effect is not very pronounced, and furthermoreis greater on ionic than on neutral samples [22]. This wasconfirmed by our own works [21]; the effect of columntemperature was hardly noticeable. Nevertheless, highcolumn temperature in combination with a flow pro-gram could be promising to significantly reduce analysistime for other methods. The use of high temperature isalso limited by the low temperature stability of somecompounds.

Conventional and monolithic columns are stable overthe same temperature range up to 458C. Elevated temper-


Figure 3. (a) Separation of a basic mixture of seven alkaloids. Column: one Chromolith Performance Rp-18e; eluent: phosphatebuffer 25 mM, pH 3/methanol (80:20); flow rate: 3 mL/min; detection wavelength, 214 nm. Compounds are: 1, codeine phos-phate; 2, ephedrine HCL; 3, theophiline ethylenediamine; 4, atropine sulfate; 5, yohimbine HCl; 6, butylscopolamine bromide; 7,papaverine HCl; 6a, butylscopolamine impurity a; 6b, butylscopolamine impurity b. (b) Separation of a basic mixture of sevenalkaloids. Column: two connected Chromolith Performance Rp-18e; eluent: phosphate buffer 25 mM, pH 3/methanol (80:20);flow rate: 3 mL/min; detection wavelength 214 nm, peak names as in Fig. 3a. (c) Separation of a basic mixture of seven alkaloids.Column: two connected Chromolith Performance Rp-18e; eluent: phosphate buffer 25 mM, pH 3/methanol (80:20), using theabove detection wavelength 214 nm, using the flow program shown in Table 4. Peak names as in Fig. 3a.


ature is problematic with monolithic columns becausethey are packed in an insulating material of PEEK (poly-ether ethyl ketone). At least it is better to work under con-stant temperature to maintain constant retention andresolution especially for ionic samples.

A possible solution for this problem is to heat themobile phase in a water bath and to cover the mobilephase lines with insulator. At the same time, a stainlesssteel capillary connected to the column inlet should beplaced with the column in the oven. This will insure acertain temperature inside the column in spite of theinsulating column tube. Furthermore, other heatingstrategies as microwaves could be tried in column ovens.

In addition to the above discussed parameters, havinga high porosity and low backpressure offer the advan-tages of decrease in washing and re-equilibrium timesfor monolithic columns during method development.This could be done by applying high flow rates for re-equilibrium and washing up to 9 mL/min for 3 min (seealso Chart 1). Furthermore, this eliminates columnblockage by contaminants.

As the density of monolithic columns is much lower,the loadability of a conventional column of the same sizeis much higher [26]. Accordingly, one should take carenot to inject a too large sample weight or volume as it ispossible for the mass of the sample to overload the col-umn.

Real resolution (also efficiency) is decreased on increas-ing the flow rate [27]. The peak height and thus sensitiv-ity are not increased in contrast to the mobile phase gra-dient elution. The peak widths are compressed only intime but not in volume.

A slight drawback of flow programming is the morecomplicated quantification using the peak information.Quantification in HPLC is usually based on the assump-tion that time is proportional to mobile phase volume.This is no longer valid when using a flow rate gradient.

Preliminary study shows that within-run repeatabilityis not compromised using flow programming comparedto isobaric conditions [21]. However, when comparingpeak areas within a run, the flow program has to be con-sidered. All peak areas have to be corrected by the effec-tive flow rate when the analytes pass the detector cell.This is similar to the situation in CE where correctedpeak areas are used. This measure is obtained by dividingthe peak areas by the corresponding migration times[28].

Similarly, corrected areas could be obtained using theeffective flow rate (Eq. 1):

AC = Afv,e (1)

Here Ac is the corrected peak area, A is the actual peakarea, and fv,e is the effective flow volume.

From our investigations applying conventional Super-spher and Chromolith Performance C18 columns in pilo-carpine, propranolol, glibenclamide, glimepiride, andinsulin methods of analysis, we came to the conclusionthat repeatability was slightly better on monolithic col-umns than on the conventional columns possibly due tothe better peak shape and reduced baseline noise thatleads to more precise integration. Furthermore, mono-lithic columns were found to provide a good column-to-column reproducibility [27].

5 Conclusion

The most important advantages of developing methodswith monolithic silica columns include the ability tosave time for finding initial separation conditions or fur-ther optimization of selected conditions due to theapplicability of high flow rates and so shorter run times.Monolithic columns also provide rapid washing andequilibrium times. The application of flow programmingon monolithic columns is beneficial for further reduc-tion in the analysis time. Monolithic columns have alsoshown to be advantageous in developing methods forcomplex mixtures by connecting two or more columnstogether to increase the separation efficiency, withoutunduly prolonging the analysis time.

I would like to thank Mr. L. Kaminski and Mr. A. Wittneben fortheir commitment in the analysis of the basic alkaloid mixture.

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a strategy to develop fast rp-hplc methods using monolithic silica columns

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