Electrophoretic Systems Separation Strategies

Analysis of native amino acids Direct UV detection at wavelengths below 220 nm takes advantage of the absorptivity of the carbonyl bond. Detection at such nonspecific wavelengths requires highly transparent buffers. Borate and phosphate are convenient electrolyte systems. Selectivity is mainly achieved by the optimization of pH because the analysis is performed with the native species.

In order to obtain cationic analytes, pH has to be adjusted to values lower than the first dissociation step (pK & 2). The stability of fused-silica capillaries is restricted to pH values above 2.5. Thus basic conditions with analytes migrating counter to the EOF are preferred. This separation mode benefits by prolonging the effective separation distance, keeping the electrical field strength constant so that higher resolution is achieved. Limits of detection are in the range of about 10"4molL" (Figure 1).

Figure 1 Separation of amino acids and dipeptides in an infusion solution using direct detection at low wavelength. Capillary: fused silica 75 |im i.d., 65/73.5 cm; buffer: borate 40 mmol L"1, pH = 11.0; E = 408 Vcm"1,191 nm; injection 50 mbar, 5 s. 1, Lys; 2, Pro; 3, Try; 4, Leu; 5, Ile; 6, Gly-Glu; 7, Val; 8, Phe; 9, His; 10, Met; 11, Ala; 12, Thr; 13, Ser; 14, Gly-Tyr; 15, Glu; 16, Asp.

Figure 1 Separation of amino acids and dipeptides in an infusion solution using direct detection at low wavelength. Capillary: fused silica 75 |im i.d., 65/73.5 cm; buffer: borate 40 mmol L"1, pH = 11.0; E = 408 Vcm"1,191 nm; injection 50 mbar, 5 s. 1, Lys; 2, Pro; 3, Try; 4, Leu; 5, Ile; 6, Gly-Glu; 7, Val; 8, Phe; 9, His; 10, Met; 11, Ala; 12, Thr; 13, Ser; 14, Gly-Tyr; 15, Glu; 16, Asp.

Indirect UV detection was evolved for the analysis of small inorganic ions but it is also an efficient technique for analysis of a broad range of nonabsor-bing components. This methodology is performed very easily with CE using a UV-absorbing electrolyte. With respect to dissociation behaviour, mobility and absorptivity the background electrolyte (BGE) has to be chosen carefully. As mentioned above, basic conditions should be applied to generate anionic species of amino acids. Therefore the BGE has to be negatively charged under alkaline conditions. Beside generating the background signal the nature of the electrolyte used has great influence on separation selectivity. Best resolution can be achieved with electrolytes of moderate mobility, e.g. salicylic acid (|pH = 11.5 = — 6 x 10~4 cm2 V-1 s"1). Salicylate at low mmolL"1 concentrations may also be used for indirect fluorescence detection. Concentration limits are in the range of 10_5 mol L"1 (Figure 2).

Another approach to a universal, high sensitivity detection scheme is mass spectrometry (MS). Beside the very low limits of detection which are achievable, this technique provides information about molecular mass and structure. The compatibility of capillary zone electrophoresis (CZE) to MS can be attributed to the low flow rates in CZE. The main problem in coupling CZE to MS is the buffer. Further developments on suitable volatile buffers and interface types will extend the scope of applications.

Analysis of derivatized amino acids Many of the chemical reactions for labelling originate from pep-tide synthesis where they were used as protective groups or sequencing agents.

As a consequence of derivatization, amino acids change from small ionic species to large hydrophobic molecules. Differences in mobilities decrease. A sufficient separation selectivity is mainly achieved by micellar electrokinetic capillary chromatography (MEKC).

Many reagents have been investigated to improve sensitivity as well as suitability for fluorescence detection. Depending on the separation problem, further requirements have to be considered. The reagent must react quantitatively and reproducibly with primary and secondary amines to form stable products. Side reactions and fluorescence of the tag itself can interfere with the analysis. The choice of derivatizing agent is limited by these prerequisites.

The commonest applied systems are discussed below (Figure 3).

The classical agent ninhydrin is not used for de-rivatization in CE because the aldehydes formed cannot be separated.

O-phthaldialdehyde (OPA) was one of the first reagents developed for pre-column derivatization in liquid chromatography (LC). Strongly absorbing isoindoles with fluorescence properties are formed in a rapid reaction. The stability of the derivatives mainly depends on the amino acid and the reducing agent, e.g. thiols. Unfortunately, secondary amines are not derivatized. An increase in stability and detection sensitivity has been achieved by using naphthalene-2,3-dicarboxaldehyde (NDA) or 3-(4-carboxyben-zoyl)-2-quinolinecarboxaldehyde (CBQCA).

Phenylthiohydantoins (PTH) of amino acids are generated during Edman degradation of peptides. Maximum absorbance is found at 254 nm but the

Figure 2 Separation of amino acids and dipeptides using indirect detection. Capillary: fused silica 75 |im i.d., 86.5/95 cm; buffer: salicylic acid 5 mmol L_1; pH = 11.5; E = 316 Vcm~1, 214 nm; injection 50 mbar, 5 s. 1, Lys; 2, Pro; 3, Try; 4, Gly-Glu; 5, Leu; 6, Ile; 7, Val; 8, His; 9, Met; 10, Ala; 11, Thr; 12, Asn; 13, Ser; 14, Gly; 15, Tyr; 16, Ac-Tyr; 17, Cys-Cys; 18, Ac-Cys; 19, Glu; 20, Asp.

Figure 2 Separation of amino acids and dipeptides using indirect detection. Capillary: fused silica 75 |im i.d., 86.5/95 cm; buffer: salicylic acid 5 mmol L_1; pH = 11.5; E = 316 Vcm~1, 214 nm; injection 50 mbar, 5 s. 1, Lys; 2, Pro; 3, Try; 4, Gly-Glu; 5, Leu; 6, Ile; 7, Val; 8, His; 9, Met; 10, Ala; 11, Thr; 12, Asn; 13, Ser; 14, Gly; 15, Tyr; 16, Ac-Tyr; 17, Cys-Cys; 18, Ac-Cys; 19, Glu; 20, Asp.

Figure 3 Structures of derivatizing reagents. OPA, o-Phthalaldehyde; NDA, naphthalene-2,3-dicarboxaldehyde; CBQCA, 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde; PITC, phenylisothiocyanate, DNS, 5-dimethylaminonaphthalene-1-sulfonyl chloride; DABS, dimethylaminoazobenzenesulfonyl chloride; FMOC, 9-fluorenylmethyl chloroformate; FLEC, (R (S)-1-(fluorenyl) ethyl chloro-formate.

Figure 3 Structures of derivatizing reagents. OPA, o-Phthalaldehyde; NDA, naphthalene-2,3-dicarboxaldehyde; CBQCA, 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde; PITC, phenylisothiocyanate, DNS, 5-dimethylaminonaphthalene-1-sulfonyl chloride; DABS, dimethylaminoazobenzenesulfonyl chloride; FMOC, 9-fluorenylmethyl chloroformate; FLEC, (R (S)-1-(fluorenyl) ethyl chloro-formate.

derivatives lack fluorescence. Analysis is performed using phosphate or borate buffers under alkaline conditions. Surfactants such as sodium dodecyl sulfate (SDS) give a micellar pseudo-stationary phase allowing the partition process. In contrast to cationic surfactants, e.g. dodecyltrimethylammonium bromide (DTAB), analytical systems using anionic surfactants benefit from a wider migration time window. This can be mainly attributed to their counterosmotic migration behaviour (Figure 4).

Sulfonyl chlorides can convert primary as well as secondary amines. Well-known representatives are dansyl (DNS) and dabsyl (DABS) chloride. In order to separate all DNS amino acids, acidic buffers are used to reduce the EOF. In addition, neutral surfactants such as TWEEN 20 have been applied. The main disadvantage is the prolonged analysis time of about 70 min. Faster separations can be achieved using SDS with the penalty of a decrease in resolution. In some cases resolution can be enhanced by operating at lower temperatures (Figure 5).

Carbonyl chlorides such as fluorenylmethyl chloroformate (FMOC) are more reactive than sulfonyl chlorides. FMOC amino acids fluoresce strongly and are stable at room temperature. Detection sensitivities in the nmol L"1 range can be achieved.

Beside fluorescence detection, further improvements in sensitivity and specificity can be obtained with laser-induced fluorescence (LIF) techniques. A prerequisite is the match of emission wavelengths of the derivatized analyte with the spectral lines of the lasers. Great effort has been invested in the development of new fluorophores such as TRTC, CTSP, TBQCA, IDA and CBQ (Table 1).

Unfortunately, most of them are not commercially available.

Different derivatization techniques are applied: pre-column tagging is the commonest method. Several attempts have been made to transfer post-column methodology from LC to CE. A further promising technique is derivatization in the capillary because it simplifies automation. Reagent and sample are injected in succession. With the tandem mode a plug of reagent is injected into the column followed by the sample. A second technique is the introduction of an additional plug of reagent after the sample (sandwich

Figure 4 Separation of 20 PTH amino acids by MEKC. Capillary: fused silica 50 ^m i.d., 59/67.5 cm, buffer: phosphate 25 mmol L 1; SDS25 mmol L"1; pH = 9.0; E = 444 Vcm"1, 260 nm; injection 50 mbar, 5 s. 1, Thr; 2, Asn;3, Ser; 4, Gln;5, Asp; 6, Gly; 7, Ala; 8, His; 9, Glu; 10, Tyr; 11, Cys; 12, Pro; 13, Val; 14, Met; 15, Leu; 16, Ile; 17, Try; 18, Phe; 19, Lys; 20, Arg.

Figure 4 Separation of 20 PTH amino acids by MEKC. Capillary: fused silica 50 ^m i.d., 59/67.5 cm, buffer: phosphate 25 mmol L 1; SDS25 mmol L"1; pH = 9.0; E = 444 Vcm"1, 260 nm; injection 50 mbar, 5 s. 1, Thr; 2, Asn;3, Ser; 4, Gln;5, Asp; 6, Gly; 7, Ala; 8, His; 9, Glu; 10, Tyr; 11, Cys; 12, Pro; 13, Val; 14, Met; 15, Leu; 16, Ile; 17, Try; 18, Phe; 19, Lys; 20, Arg.

mode). After a specified time for reaction, the separation can be performed.

Chiral analysis Assays of enantiomeric purity are easily performed by CE by simply adding the chiral selector to the running buffer. Two different methodologies are applied to achieve resolution. First, chiral distinction can be established by the formation of non-covalently bonded diastereomers.

The most widely applied cyclodextrins form host-guest complexes with one of the enantiomers preferentially. Compared to migration in the bulk phase, the complexed species possesses a different mobility. The separation occurs due to different complex stabilities resulting in different migration velocities. Enhancement of enantioselectivity is primarily attributed to cavity size (a-, ft-, y-cyclodextrin (CD)) and derivatization of the hydroxy moieties of

Figure 5 Separation of DNS amino acids by MEKC in an infusion solution. Capillary: fused silica 50 ^m i.d., 50/57.5 cm, buffer: borax 20 mmol L"1 SDS 102.5 mmol L"1; pH = 9.1; E = 435 V cm"1, 214 nm; T = 7.50C; injection 50 mbar, 5 s. 1, Thr; 2, Ser; 3, Ala; 4, Gly; 5, Glu; 6, Val; 7, Pro; 8, Met; 9, Ile; 10, Leu; 11, Phe; 12, Try; 13, Arg; 14, His; 15, Tyr; 16, Di D-Lys.

Figure 5 Separation of DNS amino acids by MEKC in an infusion solution. Capillary: fused silica 50 ^m i.d., 50/57.5 cm, buffer: borax 20 mmol L"1 SDS 102.5 mmol L"1; pH = 9.1; E = 435 V cm"1, 214 nm; T = 7.50C; injection 50 mbar, 5 s. 1, Thr; 2, Ser; 3, Ala; 4, Gly; 5, Glu; 6, Val; 7, Pro; 8, Met; 9, Ile; 10, Leu; 11, Phe; 12, Try; 13, Arg; 14, His; 15, Tyr; 16, Di D-Lys.

Table 1 Examples of derivatizing reagents and detection wavelengths

OPA 334 455

NDA 462 490

CBQCA 450

PITC 254

DNS 14100 254 570

DABS 420-450

FMOC-Cl 265 315

FLEC 265 310

TRTC > 100 000 540 567

CTSP 82 000 663 687

TBQCA 465 550

IDA 33 100 409 482

CBQ 466 544

Presence of reducing agents (thiols), strong absorbance, strongly fluorescence, unreacted OPA not fluorescent, derivatives lack of stability, reaction rapid Reaction rapid, increased stability compared to OPA, recently commercially available

Peptide sequencing by Edman degradation, cyclic thiohydantoins; no fluorescence properties Problems with derivatization by-products

Fluorogenic derivatives with primary/secondary amines, strong absorbance

Converts enantiomers to diastereomers

Ex at 540 nm matches with emission line of low cost HE

laser

Semiconductor laser

OPA, o-Phthalaldehyde; NDA, naphthalene-2,3-dicarboxaldehyde; CBQCA, 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde; PITC, phenylisothiocyanate; DNS, 5-dimethylaminonaphthalene-1-sulfonyl chloride; DABS, dimethylaminoazobenzenesulfonyl chloride; FMOC, 9-fluorenylmethyl chloroformate; FLEC, (R) (S)-1-(-fluorenyl)ethyl chloroformate; TRTC, tetramethylrhodamine isothiocyanate; CTSP, pyronin succinimidyl ester; TBQCA, 3-(4-tetrazolebenzoyl)-2-quinolinecarboxaldehyde; IDA, 1-methoxycarbonylinodolizine-3,5-dicarbaldehyde; CBQ, 3-(p-carboxybenzoyl) quinoline-2-carboxaldehyde.

the cyclodextrin (methyl-, hydroxypropyl-, sul-fobutyl-CD). Whereas compounds with a single aromatic core fit into a-CDs, ^-CDs mainly form complexes with polynuclear aromates such as tyr or try. Larger structures are accommodated by y-CDs. Most of the enantiomeric separations are performed using phosphate or borate electrolytes with native or y-CD or mixed MEKC-CD systems which additionally contain a surfactant, mostly SDS.

Additives like urea or small amounts of organic solvents can improve the resolution.

Chiral surfactants such as N-dodecanoyl-l-serine (SDVal) or N-dodecanoyl-l-glutamate (SDGlu) have been investigated. These amino acids with hydropho-bic alkyl chains are applied in a mixture with nonchiral surfactants, e.g. SDS.

Metal ions of copper(II), zinc(II) or cobalt(III) can be added to the electrolyte containing an l-isomer of an amino acid, e.g. l-proline, l-histidine or a dipep-tide, e.g. aspartame. These metal-amino acid or metal-dipeptide complexes preferentially form a ternary complex with one enantiomer of the amino acid in the sample. Separation occurred due to different complex stabilities resulting in different mobilities for the individual enantiomers.

As a second approach, a racemic mixture of amino acids is derivatized with an optically pure reagent yielding covalent-bonded diastereoisomers. Reagents like GITC (2,3,4,6-tetra-O-acetyl-^-d-glucopyran-

osyl isothiocyanate) allow the application of non-chiral separation techniques. Detection sensitivity can be improved simultaneously by using reagents like FLEC ((R) or (S)-(1-fluorenyl)ethyl chloroformate) containing chromophores or OPA with a chiral thiol.

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