750
Electrophoresis 1990, 11, 750-756
F. S. Stover
Frederick S. Stover Monsanto Chemical Company, Detergents and Phosphates Division, St. Louis, MO
Applications of capillary electrophoresis for industrial analysis Capillary electrophoresis techniques, particularly isotachophoresis and zone electrophoresis, are useful for the determination of various analytes in a wide range of sample matrices. This paper reports applications of capillary electrophoresis developed for detergent, food additive, herbicide, animal nutrition and biotechnology samples. Major advantages of capillary electrophoresis include versatility and speed of method development. Options for small ion analysis using modern, fused silica capillary instruments are discussed.
1 Introduction Capillary electrophoresis (CE) techniques are aversatileclass of analytical separation methods based on differential migration of analyte species in narrow bore tubes [ l ] . The techniques are characterized by rapid analysis times (10-60 min), small sample sizes (10 nL-IOpL), high voltages (5-50 kV), nanogram sensitivities and high resolutions. A variety of separation mechanisms have been employed, and include free solution zone electrophoresis (CZE) I2 I, polyacrylamide gel sieving 131, micelle-modified zone electrophoresis 141, isoelectric focusing [51 and isotachophoresis (ITP) 161. In our laboratories, we have found ITP and C Z E useful for rapid analytical separations. The techniques are complementary to liquid, gas and ion chromatography for small molecule separations and to gel electrophoresis for biopolymer analvsis. ITP uses discontinuous buffer systems and is performed in 0.5-0.8 mm i.d. polytetrafluoroethylene(PTFE) capillaries with conductivity and LJV detection. C Z E normally uses a single buffer in 25- 100 pm i.d. fused silica capillaries with UV detection. These methods possess many practical advantages for industrial analysis vs. complementary separation techniques.
I n ITP, a single separation capillary can be used for all separations. By reversing the power supply polarity, either anion or cation analysis can be performed. Separation conditions and selectivities can be altered over a wide range by varying electrolyte composition. Thus, numerous conditions can be tested in a single day without concern for hardware changes and equilibration. Inertness of the ITP capillary allows injection of aggressive samples, slurries and particulates without regard to column life. Separation quality is reproducible over long periods of time. Compared to chromatographic separations, ITP is extremely selective to analyte charge type. Neutrals and ions opposite in sign to those of interest never even reach the detector. Finally, the discontinuous electrolyte system ensures relatively constant run times. Appearance of the terminating ion signals the end of the separation, and there is no need to wait for “late eluting” species. The advantage of using homogeneous buffer conditions to affect the separation also applies to CZE. Fused silica tubing, however, has shown less inertness than PTFE, and capillary Correspondence: Dr. Frederick S. Stover, Monsanto Co., 800 N. Lindbergh Blvd.. St. Louis, MO 63 167, USA
Abbreviations: CE, capillary electrophoresis: CZE, capillary zone electrophoresis; HPLC, high performance liquid chromatography; ITP, iso tachophorew ;PTFE, polytetrafluoroethylene r. VCH Verlagsgeselltchaft mbli, D 6940 Weinhelm. 19’90
conditioning between runs or when changing buffers may be needed. C Z E also is attractive from the points of view of speed and sample consumption. CZE‘s main advantages are high efficiencies and resolutions for macromolecules compared to other techniques. Both ITP and C Z E benefit frbm having their selectivities based on differences in electrophoretic mobilities. These predominantly charge-based separations offer useful alternatives to separations based on size or hydrophobicity. C E separations in open capillaries are well-described by fundamental migration and homogeneous equilibrium equations, and excellent prediction of separations can be performed if a few physico-chemical constants are known 171. This paper reports a variety of applications of C E techniques to practical problems in industrial chemical analysis. The applications presented here are meant to highlight the utility of C E in a modern instrumental analysis laboratory.
2 Materials and methods An LKB 2 127 Tachophor (Bromma, Sweden) was used for all electrophoresis experiments. In theITP mode, a 200 x 0.8 mm PTFE capillary was connected to an LKB 2127-140conductivity/UV detector. Data was acquired using a strip chart recorder or a microcomputer-based system described earlier I8 I. Electrolyte compositions and operating parameters are described below for individual separations. C Z E was performed on a Tachophor modified for fused silica capillary operation, as described previously [91. An ISCO (Lincoln, NE, USA) V 4 UV-VIS detector was employed at 0.01 AUFS. Chemicals used for electrolytes, buffers and as standards were commercial reagent-grade materials and were used without further purification. Table I lists the composition of some common ITP electrolytes and C Z E buffers used here.
3 Results and discussion 3.1 Isotachophoresis Recent advances in the theory, instrumentation and applications ofITP have been reviewed by BoEek [ 101.Baldesten gave an earlier overview of ITP’s applicability to avariety of sample types 1 11. We present here some separations developedin our laboratories which illustrate the utility of ITP.
3.1.1 Carboxylates and phosphates ITP has found wide application in the analysis of foods and beverages [ 12, 131. One advantage of ITP in this area is the 01 73-0835/90/090Y-0750 $ 3 . 5 0 ~25/0
Industrial applications of capillary electrophoresis
Electroohoresis 1990. J I . 750-756
ease of sample preparation, since simple dilution or extraction can be performed prior to direct injection. Separations are relatively insensitive to proteinaceous material or UV absorbers which might interfere with chromatographic methods. Species of interest in food and beverage analysis include carboxylic acids, which are readily determined by ITP [141. Figure 1a shows a separation of 12 common carboxylates in a pH 3.3 buffer obtained in 20 minutes. Condensed phosphates are a second class of compounds easily separated by ITP [15, 161. Figure l b gives a separation of ortho- through trimetaphosphate in p H 3.3 electrolytes. Applications of ITP to food analysis in our laboratories have included determination of organic acids and phosphates in soft drink mixes, analyses for acidulents in cheese products and phosphate speciation in powdered baking mixes. In the latter application, simple extraction with dilute HCI can be used prior to injection
,/
oxalate maleate formate glyoxylate
CI-
b -.
trimetatripolyPYro-
\
Lortho-
--- glycolate lactate
7
gluconate benzoate
I
acetate
cond.
I!
75 1
without filtration. Sample components such as whey, flour, egg proteins, carbonates and enzymes have no deleterious effect on the separation.
3.1.2 Aminoalkylphosphonic acids
ITP is well suited to the identification and quantitation of aminoalkylphosphonic acids (AAPA’s) as shown in our I I 7 I and other laboratories 1181. These polybasic acids find use in scale inhibition and corrosion control. While solutions of AAPA’s can be studied by NMR or ion chromatography, we have found ITP to be an accurate and simple alternative. ITP also excels at determining impurities such as phosphite, orthophosphate, acetate and formate. Separations of AAPA’s and related ions using standard and metal-modified electrolyte systems are shown in Fig. 2. Commercial phosphonates nitrilotri(methy1enephosphonic) acid (NTP), hydroxyethylidenedi(methy1enephosphonic) acid (HEDP), ethylenedi aminetetra(methy1enephosphonic) acid(EDTP),hexamethylenediaminetetra(methy1enephosphonic)acid (HDTP) and diethylenetriaminepenta(methy1enephosphonic) acid (DTPP) can be separated in either system. Standard pH 6 electrolytes are excellent for quantitation of single phosphonates and impurities. For mixtures of phosphonates, addition of 2 mM Zn affords better resolution through metal-ligand interactions. Metal complexation will usually reduce anionic electrophoretic mobilities, and differences in metal binding strength can be exploited to differentially alter mobilities.
propionate
caproate
7 -
T - I - - - r 17
18
19 rnl”
17
18
19 rnm
Figure 1. Conductivity signals from ITP separations of (a) carboxylates and (b) phosphates using pH 3.3 electrolytes from Table I . Separation current: 200 pA for 15 min, detection current 50 FA. Approximately 0.2 pgof each carboxylate and 0.5 pg of each phosphate injected.
We have used ITP for numerous phosphonate analyses, in cluding (1) determination of N T P electrolysis products I 191, (2) determination of phosphonate thermal stabilities and degradation products, (3) analysis of boiler waters, automobile radiator fluids and detergents for phosphonate content and (4) determination of NTP in dosed rat feeds. ITP is clearly superior for the latter analyses where polyvalent metals extracted from the feed can poison chromatographic columns.
Table 1. Composition of common electrolytes and buffers used for ITP and C Z E
pH 3.3
pH 6.0
pH 8.8
Leading ion Leading counter ion Leader additive Terminating ion Terminating counter ion Terniinatine pH
10 ItlM HCI @- Alanine 0.2 % HPMCh) 10 m M Caproic acid
10 m M HCI L-histidine 0.2 % HPMCh) 10 mM MESCl Trisd) 6.0
10 mM HCI Ammediol”) 0.2 % HPMCbl 10 mM p-Alanine Ba(OH),
11 P caiion electrolytes Leading ion Leading counter ion Terminating ion Terminating counter ion Termin:itine p H
PH 10 mM HCI
ITP anion electrolytes ~~~
C Z t buffers
~
~~~
-
=
-
10 mM Tris
___
pH 4.5 10 mM KOAc HOAc 10 mM HOAc
HCI 8.5
-
pH 7.5
pH 9.0
20 mM Tricinee) 20 mM C H E S 10 mM KCI 10 mM KCI (adjusted to proper pH with 1 M KOH)
a) Ammediol, 2-amino-2-methyl-1,3-propanediol b) HPMC, hydroxypropylmethyl cellulose c) MES, 2-(N-morpholino)-ethane sulfonic acid d) Tris, tris(hydroxymethy1)aminomethane e) Tricine, N-tris(hydroxymethy1)-methylglycine f) CHES. 2-(N-cyclohexylamino)-ethanesulfonic acid
9.0
752
F. S. Stover
Electrophoresis 1990,11, 750-756
+diff.
cond.
I
I
I
14
15
16 min.
: 16 min. 14
ITP will generally dissociate all but the most stable metalligand complexes in the samples [20], particularly using low pH electrolytes. In the above method developments, simple dilution and injection were sufficient to quantitate phosphonates, and no interference was seen when strong acids, glycols, polyacrylates or surfactants were present in the samples.
ct-
1 \
15
Another important phosphonate compound is N-phosphonomethylglycine, or glyphosate, the active ingredient in Roundup@herbicide. ITP separations of this and other herbicides have been described previously I2 1,221. Figure 3 shows our ITP separation of glyphosate and related compounds. Addition of Ca2+ to pH 6 electrolytes improves the separation through metal complexation effects. Analyses for glyphosate have been developed for synthetic samples, plant matter extracts and process streams.
3.1.3 Amino acids and analogs
HCOOH
glyp hosate
1
ITP separations of amino acids suffer from poorer resolutions and sensitivities compared to high performance liquid chromatography (HPLC) methodology. However, nearly complete ITP separation of the 20 common protein amino acids has been obtained [ 231 and no derivatization is necessary. ITP has the advantage of being a rapid and simple method for quantitating a few amino acids and is useful when analyzing complex matrices. ITP may be preferred when other species also must be determined. For example, we have used cationic ITP to simultaneously determine K and lysine 1241. Another application from our laboratory [251 is the analysis of poultry feed for the methionine analog, 2-hydroxy4(methylthio)-butanoic acid (HMB). Figure 4 gives the conductivity record for blank and HMB-containing feeds using pH 6 electrolytes. ITP analysis was found to be faster and more sensitive than G C methods, and was less prone to interferences than HPLC. Total extraction/analysis times of 20 min and sensitivities of0.04 % HMB in feed were obtained.
L. H2OBPCHO
cond.
--r---19
Figure 2. Conductivity and differential conductivity signals from ITP separations of aminoalkylphosphonic acids using p H 6 electrolytes from Table 1 with (a) 0 mM and (b) 2 mM ZnC1, added to leader. Separation current 250 pA for 10 min, detection current 50 pA. Approximately 1 yg each aminoalkylphosphonic acid and0.5 pg phosphate and phosphite iniected. For abbreviations see Section 3.1.2. Reprinted from [ 171, with permission.
21
23 rnin.
Figure 3. Conductivity signal for the ITP separation of glyphosate and related compounds. pH 6 electrolyte from Table 1 with 2 mM CaCI, added to leader. Separation current 200 yA for 15 min, detection current 50 yA. Approximately 0.5 yg phosphite and phosphate and 1 pg other components injected.
+
3.1.4 Polyelectrolytes Polyelectrolytes are often difficult to analyze by HPLC or ion chromatography due to concerns about column poisoning and regeneration. We have found ITP in open PTFE capillaries to be an excellent alternative for polyelectrolyte
Electrophoresis 1990,11, 750-756
Industrial applications of capillary electrophoresis
a
I
--\, A
Figure 4 . ITP conductivity and differential
HMB ‘ONE
conductivity traces for (a) blank poultry feed and (b) feed containing ca. 0.2 96 2-hydroxy-4(methylthio)butanoic acid (HMB) using p H 6 electrolytes from Table 1. Injections (5 pL) 1 gram feed extracted with 10 mL water for 15 min. Reprinted from 1251, with permission.
b-.--;
j cond. t
4
CI-
CI-
-
diff.
L
cond.
CI-
753
A
fumarate
%aleate
C0,‘- + polymer ,methyl
maleate
copolymer and impurities by ITP; (a) standards and (b) 10 pg polymer in p H 8.8 electrolytes from Table 1;(c) 10 pgpolymer
P-ala
analyses. Many polyelectrolytes are mobile in 1TP and determination of both polymers and their impurities can be performed. Figure 5 shows separations obtained with p H 9 electrolytes of a partially butylated styrene-maleic acid copolymer. The polyelectrolyte interferes with determination of the methyl and butyl half esters of maleate using standard pH 9 electrolytes. Advantage can be taken ofdifferential complexation of the polymer with Ca2+(Fig.5c) to achieve separation of half esters, maleate and fumarate. The sloping conductivity trace seen in Fig. 5b is not uncommon in polyelectrolyte separations, and may indicate microheterogeneity of the polymer. In addition to monomer analysis, determination of polyelectrolytes themselves can be performed. ITP has been used in our laboratory to determine po\yacrylates in detergent formulations. Also, we have found the wide pH range available to ITP important for the determination of monomers in polycarboxylates when polymer stability is a concern. Whitlock [26,27] has used ITP to assess the degree of homogeneity, charge density and counter-ion binding in a variety of polyelectrolytes.
species - is seen. Combined UVfconductivity detection was useful for identifying 2-(sulfono)-nonanoylbenzene sulfonate and 2-(sulfono)-nonanoic acid, since standard materials were not available.
3.1.6 Cations One of the most useful applications of ITP is in the determination of cations [30, 3 11. ITP allows simultaneous determina-
t
I
CI-
cond.
7 ” I
3.1.5 Surfactants ITP electrolyte systems have been developed for the analysis of different classes of surfactants 1281. These direct methods offer minimal sample handling compared to extraction and titration methods. Trimethylalkyl ammonium cations with alkyl chain lengths up to 16 are generally mobile in potassium acetate/acetic acid (KOAc/HOAc) electrolytes. Standard p H 6 electrolytes can be used for alkylsulfates and alkylbenzene sulfonates. We have used the methanolic electrolyte systems reported in the literature [29] for the determination of fatty acids in formulated soaps. In some cases, chain length discrimination by ITP is poor. This can be exploited, however, to yield simple class separations since response factors of homologs are similar. Figure 6 shows conductivity and UV signals from the separation of an alkanoyloxybenzene sulfonate synthesis mixture using p H 6 electrolytes. A good separation - based on number of acid groups and size ofeach
I
I
I
I
9
11
13
15 min.
Figure 6 . Separation of 10 pg of a partially purified surfactant synthesis mixture by ITP using p H 6 electrolytes from Table 1. Conductivity signal and UV signal at 254 nm. (1) Sulfuric acid, (2)2-(su1fono)-nonanoic acid, (3) 2-(sulfono)-nonanoyl-p-benzenesulfonic acid, ( 4 ) p-phenol sulfonic
acid, ( 5 ) unknown impurity and (6) nonanoyl-p-benzene sulfonic acid.
754
Electrophoresis 1990,lI, 750-756
F. S. Stove1
'I
Figure 7. Conductivity signals from separations of alkali metals and ammonia in pH 2 cation electrolytes from Table 1 with (a) 0 mM and(b) 5 0 m 18-crown-6 ~ ether added. Reprinted from [241, with permission.
b t i
Rb NH44-K
cond.
1 7 8
10
12 min.
9
13 min.
11
tion of both inorganic and organic bases, often with little method development. We have found many organic bases to be mobile in KOAc/HOAc electrolytes 1321. Cationic ITP applications in our laboratory include quantitating K', Na', NH, +, Ca", trimethyl-, tributyl- and isopropylammonium, guanidine and substituted guanidinium cations, trimethylsulfonium, capryltrimethylammoniuni, para- and diquat, aminotriazole, TRlS, triethanolamine and morpholine in a variety of samples. Neutral ionophores have shown considerable utility for tailoring ITP separations 133, 341. Figure 7 shows a separation developed in our laboratory for alkali metal cations with and without added 18-crown-6 ether 1241. Fifty mM 18-C-6 allows complete separation of alkali metals, while 3 mM 18-C-6 is sufficient for obtaining NH, L/K+separations
3.2 CZE C Z E has shown considerable promise for high resolution analytical 1401 and preparative 1411 separations. C Z E is particularly well-suited to analysis of biological macromolecules 142-441, where theory predicts very high theoretical plate numbers due to small diffusion coefficients. In our laboratories, C Z E has been tested for separations involving peptides and proteins.
a
CI -
b
"'
i
3.1.7 Physical constant determinations C E methods are attractive for the determination of physical constants such as ionic mobilities or single ion conductances, pKa's and metal-ligand binding constants. Hirokawa and Kiso (35-391 have shown the utility of JTP for determining these constants. Advantages of ITP (and CZE) for physical chemistry studies include applicability to niicro samples and on-line purification prior to measurement. ITP was employed when we needed to determine the limiting single ion conductance of glyphosate in order to interpret salt conductivity data. Normal conductance methods were difficult to use with this anion due to its polybasic nature. By employing a pH 4.1 electrolyte system, glyphosate could be buffered predominantly to its monovalent form. ITP runs of glyphosate anion and isopropylammonium (IPA) cation are shown in Fig. 8. Bromate and potassium were used as internal mobility standards for the anion and cation runs, respectively. Using formulas deveioped by Hirokawa and Kiso [351, single ion conductances of glyphosate and IPA were found to be 26.9 and 39.3 S-cm2/eq,respectively. IP A's single ion conductance was checked by independent conductivity measurements and excellent agreement (39.8 S-cmz/eq) was seen.
I I
1 K'
L?
glyphosate
cond.
, 10
11
12
min
13
8
9
10
11
min
Figure 8. Conductivity signals from determinations of limiting single ion conductances (mobilities). (a) Injection of ca. 3 pg each glyphosate and sodium bromate. Electrolytes: leader 10 mM HCI + 5-aminocaproic acid, pH 4.1, leader additive 0.2 % HPMC, terminator 10 mM caproic acid. (b) Injection of 1.5 pg isopropylammonium (IPA) chloride and 1.0 pg KCI in pH 2 cation electrolyte from Table 1.
Electrophoresis 1990,1I, 750-756
155
Industrial applications of capillary electrophoresis
3.2.1 Peptides We have shown that C Z E is capableofhigh resolution separations of biologically important peptides, such as angiotensins I, I1 and I11 [91. In exploring the C Z E behavior of cationic peptides in bare fused silica capillaries, we were interested to see if metallation, as well as protonation, equilibria could be used to enhance separations. Figure 9 shows the C Z E separation of three blocked heptapeptides with one (M), two (D) and three (T) histidines in a p H 7.5 buffer. Without added metal, histidines are mostly deprotonated and the three peptides comigrate. Addition of 1 mM Zn*+allows separation of the peptides through differential metal binding. This separation is comparable to ligand exchange HPLC on immobilized-metal affinity columns. The expected migration order (T > D > M) was not seen in these experiments due to interactions of highly charged Zn-T complexes with the capillary surface. Such interactions will complicate the use of bare silica capillaries for physical constant determinations, and more inert capillaries may be needed. However, Lauer 1451 has given correlations between C Z E mobility and peptide charge and chain length.
separated in a pH 9 buffer. Very high resolutions are obtained as seen by theoretical plate numbers in excess of 100 000. Another important class of proteins are human interleukins. Interleukin-3 is one of a number of colony stimulating factors that aid in bone marrow replication. We have used C Z E to monitor recombinant human interleukin-3 (rh 1L-3)levels in a commercial preparation [471, where human serum albumin is used as a carrier (Fig. 11). Recent work 1481 has shown that nanogram quantities of rh IL-3 can be collected from a C Z E capillary and identified by amino acid sequencing or sodium dodecyl sutfate (SDS)-gel electrophoresis.
I --
0
3.2.2 Proteins C Z E separations involving proteins have been prominent in the literature [46]. Two separations developed in our laboratory are shown in Figs. 10 and 11. Figure 10 shows the separation of two different somatotropins (growth hormones) by CZE. Porcine and bovine somatotropin can be quickly
porcine
bovine
_
5
_7
~
-
~ - *
10
15
-_ min
Figure 10. CZE separation of bovine and porcine somatotropins in pH 9 buffer from Table 1. Protein solutions (4 mg/mL) were injected at 5 kV for 5 s. Separation voltage 15 kV. capillary i.d. 75 pm, length to detector 70cm, overall length 100 cm. UV detection at 230 nm and 0 01 AUFS
a
UV abs.
0
2
4
6
min.
Figure 9. Separation of blocked heptapeptides: (T) Gly-His-Gly-His-GlyHis-Gly, (D) Gly-Gly-His-Gly-His-Gly-Gly and (M) Gly-Gly-Gly-HisGly-Gly-Gly, by CZE using pH 7.5 buffer from Table 1 with (a) 0 mM and (b) 1 mM Zn(CIO,), added. Voltage 15 kV, capillary i.d. 75 pm, length to detector 40 cm, overall length 70 cm. UV detection at 220 nm and 0.01 AUFS. Reprinted from 191, with permission.
I
I
0
5
I
10 min.
Figure 11. CZE separations of (a) recombinant human interleukin-3 (rhIL-3), (b)human serum albumin (HSA)and(c)commercial rhIL-3 containing HSA. Separation conditions the same as Fig. 9 except using pH 9 buffer from Table 1. Reprinted from [471, with permission.
756
F. S. Stover
4 Concluding remarks The applications of C E presented here are intended to highlight the versatility of these separation methods for industrial analysis. From our perspective, the great strength of these techniques lies in their ability to rapidly develop analysis methods. This ability derives not only from flexible operating conditions which can be quickly varied. but also from the hardiness of the separation compartment and minimal sample preparation requirements. In our laboratories, any sample type can be tested under any electrolyte condition without fear of costly equipment replacement or down-time.
An increased emphasis on CZE for small molecule separations is expected with the advent of commercial C E instrumentation using microbore silica capillaries. When developing C Z E separations for small molecules, it would be wise not to overlook the wealth of information on mobilities, buffer systems, complexation phenomena and solvent effects developed over the last 15 years by researchers in ITP. T o date, relatively few C Z E analyses have been developed for small molecule separations. Most of these applications involve use of derivatives [49] or specific detectors [501. The current lack of universal detection, which should take full advantage of the versatility of the technique, limits applications. One approach has been the use ofindirect detection [ 5 1-531, but this method is not without limitations regarding sensitivity and dynamic range. An appealing alternative in sub-100 pm C E separations is operation in the ITP mode. Here, lack of universal detection is not as great aproblem, since the detector can serve qualitatively to mark zone boundaries. ITP separations with UV-absorbing impurities as markers already have been shown using fused silica instrumentation [ 541. The indirect-UV method of Reijenga et al. [551 for ITP also should be useful for universal detection in modern instruments. Finally, greatly increased sensitivities and load capacities may be possible through capillary coupling [561 to microbore fused silica. These factors indicate that the ITP mode may remain the method of choice for developing small molecule separations. Received December 14, 1989
5 References Jorgenson, J. W., Anal. Chem. 1986,58,743A-760A. Gordon, M. J., Huang, X., Pentoney, S. L. and Zare, R. N., Science 1988,242,224-228. Karger, B.L. and Cohen, A. S . , J .Chromatogr. 1987,397,409-4 17. Terabe,S.,Otsuka,K. andAndo,T.,Anal. Chrvn. 1985,57,834-841. Hjerttn, S. and Zhu, M.-D., J. Chromatogr. 1985,346,265-270. Everaerts, F. M., Beckers, J. L. and Verheggen, T. P. E. M., Isotachophoresis - Theory, Instrumentation and Applications, Elsevier, Amsterdam 1976. Hirokawa, T., Takemi, H., Kiso, Y., Takiyama, R., Morio, M., Fujii, K. and Kikuchi, Y., J. Chromatogr. 1984,305,429-437. Stover, F. S., Deppermann, K. D. and Grote, W. A., J . Chromatogr. 1983,269, 198-201. Stover, F. S., Haymore, B. L. and McBeath, R. J., J. Chromatogr. 1989,470,241-250. BoEek, P., Gebauer, P., Dolnik, V. and Foret, F., J . Chromatogr. 1985,334, 157-195. Hjalmarsson, S.-G. and Baldesten, A,, CRC Crit. Rev. Anal. Chem. 1981,11,261-352. IP Data Sheet CA 198-901, Application to Foodlndustry, Shimadzu Corp., Tokyo, Japan.
Electrophoresis 1990, 11, 750 756
(131 LKB Application Note 79-01 - 1 9, LKB-Produkter AB, Bromrna, Sweden. 1141 BoEek, P., Lekova, K., Deml, M. and Janak, J.,J. Chromatogr. 1976, 117,97-104. 1151 Yagi, T., Kojima, K., Nariai, H. and Motooka, I., Bull. Chem. Soc. Jpn. 1982,55, 1831-1833. 1161 Motooka, I., Nariai, H., Nakazaki, K. and Tsuhako, M., J . Chro matogr. 1984,295,533-536. 1171 Stover, F. S. and Wagenknecht, J . H., Anal. Chim. Acta 1982,135, 347-350. 1181 Fitzgerald, E. A.,J. Chromatogr. Sci. 1983,21, 188-189. [I91 Wagenknecht, J. H., Stover, F. S. and Wagner, W. G., Synth. React. Inorg. Met.-Org. Chem. 1982,12, 1-9. I201 Gebauer, P., BoEek, P., Deml, M. and Janak, J.,J.Chromatogr. 1980, 199,81-94. 1211 Kiivankova, L. and BoEek, P., Electrophoresis 1986, 7, 100-103. i221 Stransky, Z., J. Chromatogr. 1985,320,219-231. I231 Hirokawa, T., Gojo, T. and Kiso, Y., J . Chromatogr. 1986, 369, 59-81. 1241 Stover, F. S., J. Chromatogr. 1984,298,203-210. [251 Vinjamoori, D. V. andschisla, R. M.,J.Assoc. 0ff.Anal. Chem. 1986, 69,474-471. [261 Whitlock, L. R., J . Chramatogr. 1986,363,267-276. L271 Whitlock, L. R. and Wheeler, L. M., J. Chromatogr. 1986, 368, 125-134. 1281 Asakawa, T., Takeda, H., Miyagishi, S. andNishida,M.,J.Amer. Oil Chem. Soc. 1986,63, 1479-1482. 1291 Koval, M., Kaniansky, D., Hutta, M. and Lacko, R., J . Chromatogr. 1985,325,151-160. 1301 BoEek, P. and Foret, F.,J. Chromatogr. 1984,313, 189-222. 1311 Everaerts, F. M., Verheggen, T. P. E. M., Reijenga, J. C., Aben, G. V. A.,Gebauer,P. andBoCek,P.,J. Chromatogr. 1985,320,263-268. [321 Stover, F. S., J. Chromatogr. 1989,470, 201-208. 1331 Tazaki,M.,Takagi,M. and Ueno, K., Chem. Lett. 1982,639-640. I341 Snopek, J., Jelinek, I. and Smolkova-Keulemansova, E., J . Chromatogr. 1988,452,571-590. 1351 Kiso, Y. and Hirokawa, T., Chem. Lett. 1980,745-748. Kiso. Y. and Hirokawa., T.., Chem. Lett. 1980.323-326. I361 1 I371 Hirokawa, T. and Kiso, Y . , J . Chromatogr. 1982,248,341-362. [381 Hirokawa, T., Nishino, M. and Kiso, Y., J . Chromatogr. 1982,252, 49-65. [3YJ Hirokawa, T., Tsuyoshi, T. and Kiso, Y., J. Chromatogr. 1987,408, 27-41. [401 Jorgenson, J. W. and Lukacs, K. D., Science 1983,222,266-272. 1411 Rose,D. J. andJorgenson,J. W.,J. Chromatogr. 1988,438,23-34. 1421 McCormick, R. M., Anal. Chem. 1988,60,2322-2328. [431 Nielsen, R.G., Sittampalam,G.S. andRickard,E. C.,Anal. Biochem. 1989,177,20-26. 1441 Ludi, H., Gassmann, E., Grossenbacher. H. and Marki, W., Anal. Chim. Acta 1988,213,215-219. 1451 Grossman, D., Colburn, J. C., Moring, S. E. and Lauer, H. H.. First lnternational Symposium on High Performance Capillary Electrophoresis, Poster M-P-102, Boston, April 12, 1989. 1461 Lauer,H. H. andMcManigill,D.,Anal. Chem. 1986,58,166-170. 1471 Hecht, R. I., Morris, J . C., Stover, F. S.,Fossey, L. andDemarest, C., Prep. Biochem. 1989,19,201-208. 1481 Hecht, R. I., Coleman,J. F., Morris, J . C., Stover, F.S. andDemarest, C., Prep. Biochem. 1989,19, 363-367. 1491 Otsuka, K., Terabe, S. and Ando, T., J . Chromatogr. 1985, 332, 2 19-226. i501 Wallingford,R.A. andEwing,A.G.,Anal. Chem. 1988,60,258-263. I5 11 Foret, F., Fanali, S., Ossicini, L. and BoEek, P., J . Chromatogr. 1989, 470,299-308. 1521 Kuhr, W. G. and Yeung, E. S., Anal. Chem. 1988,60,2642-2646. 1531 Ewing, A. G., Wallingford, R. A. and Olefirowicz, T.M.,Anal. Chem. 1989.61.292A-303A. I541 Dionex Promotional Letter, August 8, 1989, Dionex Corporation, Sunnyvale, CA, USA. 1551 Reijenga, J. C., Lemmens, A. A. G., Verheggen, T. P. E. M. and Everaerts, F. M.,J. Chromatogr. 1985,320, 67-73. 1561 Everaerts, F. M., Verheggen, T. P. E. M. and Mikkers, F. E. P., J. Chromatogr. 1979,169, 21-38.
Electrophoresis 1990, 11, 750-756
F. S. Stover
Frederick S. Stover Monsanto Chemical Company, Detergents and Phosphates Division, St. Louis, MO
Applications of capillary electrophoresis for industrial analysis Capillary electrophoresis techniques, particularly isotachophoresis and zone electrophoresis, are useful for the determination of various analytes in a wide range of sample matrices. This paper reports applications of capillary electrophoresis developed for detergent, food additive, herbicide, animal nutrition and biotechnology samples. Major advantages of capillary electrophoresis include versatility and speed of method development. Options for small ion analysis using modern, fused silica capillary instruments are discussed.
1 Introduction Capillary electrophoresis (CE) techniques are aversatileclass of analytical separation methods based on differential migration of analyte species in narrow bore tubes [ l ] . The techniques are characterized by rapid analysis times (10-60 min), small sample sizes (10 nL-IOpL), high voltages (5-50 kV), nanogram sensitivities and high resolutions. A variety of separation mechanisms have been employed, and include free solution zone electrophoresis (CZE) I2 I, polyacrylamide gel sieving 131, micelle-modified zone electrophoresis 141, isoelectric focusing [51 and isotachophoresis (ITP) 161. In our laboratories, we have found ITP and C Z E useful for rapid analytical separations. The techniques are complementary to liquid, gas and ion chromatography for small molecule separations and to gel electrophoresis for biopolymer analvsis. ITP uses discontinuous buffer systems and is performed in 0.5-0.8 mm i.d. polytetrafluoroethylene(PTFE) capillaries with conductivity and LJV detection. C Z E normally uses a single buffer in 25- 100 pm i.d. fused silica capillaries with UV detection. These methods possess many practical advantages for industrial analysis vs. complementary separation techniques.
I n ITP, a single separation capillary can be used for all separations. By reversing the power supply polarity, either anion or cation analysis can be performed. Separation conditions and selectivities can be altered over a wide range by varying electrolyte composition. Thus, numerous conditions can be tested in a single day without concern for hardware changes and equilibration. Inertness of the ITP capillary allows injection of aggressive samples, slurries and particulates without regard to column life. Separation quality is reproducible over long periods of time. Compared to chromatographic separations, ITP is extremely selective to analyte charge type. Neutrals and ions opposite in sign to those of interest never even reach the detector. Finally, the discontinuous electrolyte system ensures relatively constant run times. Appearance of the terminating ion signals the end of the separation, and there is no need to wait for “late eluting” species. The advantage of using homogeneous buffer conditions to affect the separation also applies to CZE. Fused silica tubing, however, has shown less inertness than PTFE, and capillary Correspondence: Dr. Frederick S. Stover, Monsanto Co., 800 N. Lindbergh Blvd.. St. Louis, MO 63 167, USA
Abbreviations: CE, capillary electrophoresis: CZE, capillary zone electrophoresis; HPLC, high performance liquid chromatography; ITP, iso tachophorew ;PTFE, polytetrafluoroethylene r. VCH Verlagsgeselltchaft mbli, D 6940 Weinhelm. 19’90
conditioning between runs or when changing buffers may be needed. C Z E also is attractive from the points of view of speed and sample consumption. CZE‘s main advantages are high efficiencies and resolutions for macromolecules compared to other techniques. Both ITP and C Z E benefit frbm having their selectivities based on differences in electrophoretic mobilities. These predominantly charge-based separations offer useful alternatives to separations based on size or hydrophobicity. C E separations in open capillaries are well-described by fundamental migration and homogeneous equilibrium equations, and excellent prediction of separations can be performed if a few physico-chemical constants are known 171. This paper reports a variety of applications of C E techniques to practical problems in industrial chemical analysis. The applications presented here are meant to highlight the utility of C E in a modern instrumental analysis laboratory.
2 Materials and methods An LKB 2 127 Tachophor (Bromma, Sweden) was used for all electrophoresis experiments. In theITP mode, a 200 x 0.8 mm PTFE capillary was connected to an LKB 2127-140conductivity/UV detector. Data was acquired using a strip chart recorder or a microcomputer-based system described earlier I8 I. Electrolyte compositions and operating parameters are described below for individual separations. C Z E was performed on a Tachophor modified for fused silica capillary operation, as described previously [91. An ISCO (Lincoln, NE, USA) V 4 UV-VIS detector was employed at 0.01 AUFS. Chemicals used for electrolytes, buffers and as standards were commercial reagent-grade materials and were used without further purification. Table I lists the composition of some common ITP electrolytes and C Z E buffers used here.
3 Results and discussion 3.1 Isotachophoresis Recent advances in the theory, instrumentation and applications ofITP have been reviewed by BoEek [ 101.Baldesten gave an earlier overview of ITP’s applicability to avariety of sample types 1 11. We present here some separations developedin our laboratories which illustrate the utility of ITP.
3.1.1 Carboxylates and phosphates ITP has found wide application in the analysis of foods and beverages [ 12, 131. One advantage of ITP in this area is the 01 73-0835/90/090Y-0750 $ 3 . 5 0 ~25/0
Industrial applications of capillary electrophoresis
Electroohoresis 1990. J I . 750-756
ease of sample preparation, since simple dilution or extraction can be performed prior to direct injection. Separations are relatively insensitive to proteinaceous material or UV absorbers which might interfere with chromatographic methods. Species of interest in food and beverage analysis include carboxylic acids, which are readily determined by ITP [141. Figure 1a shows a separation of 12 common carboxylates in a pH 3.3 buffer obtained in 20 minutes. Condensed phosphates are a second class of compounds easily separated by ITP [15, 161. Figure l b gives a separation of ortho- through trimetaphosphate in p H 3.3 electrolytes. Applications of ITP to food analysis in our laboratories have included determination of organic acids and phosphates in soft drink mixes, analyses for acidulents in cheese products and phosphate speciation in powdered baking mixes. In the latter application, simple extraction with dilute HCI can be used prior to injection
,/
oxalate maleate formate glyoxylate
CI-
b -.
trimetatripolyPYro-
\
Lortho-
--- glycolate lactate
7
gluconate benzoate
I
acetate
cond.
I!
75 1
without filtration. Sample components such as whey, flour, egg proteins, carbonates and enzymes have no deleterious effect on the separation.
3.1.2 Aminoalkylphosphonic acids
ITP is well suited to the identification and quantitation of aminoalkylphosphonic acids (AAPA’s) as shown in our I I 7 I and other laboratories 1181. These polybasic acids find use in scale inhibition and corrosion control. While solutions of AAPA’s can be studied by NMR or ion chromatography, we have found ITP to be an accurate and simple alternative. ITP also excels at determining impurities such as phosphite, orthophosphate, acetate and formate. Separations of AAPA’s and related ions using standard and metal-modified electrolyte systems are shown in Fig. 2. Commercial phosphonates nitrilotri(methy1enephosphonic) acid (NTP), hydroxyethylidenedi(methy1enephosphonic) acid (HEDP), ethylenedi aminetetra(methy1enephosphonic) acid(EDTP),hexamethylenediaminetetra(methy1enephosphonic)acid (HDTP) and diethylenetriaminepenta(methy1enephosphonic) acid (DTPP) can be separated in either system. Standard pH 6 electrolytes are excellent for quantitation of single phosphonates and impurities. For mixtures of phosphonates, addition of 2 mM Zn affords better resolution through metal-ligand interactions. Metal complexation will usually reduce anionic electrophoretic mobilities, and differences in metal binding strength can be exploited to differentially alter mobilities.
propionate
caproate
7 -
T - I - - - r 17
18
19 rnl”
17
18
19 rnm
Figure 1. Conductivity signals from ITP separations of (a) carboxylates and (b) phosphates using pH 3.3 electrolytes from Table I . Separation current: 200 pA for 15 min, detection current 50 FA. Approximately 0.2 pgof each carboxylate and 0.5 pg of each phosphate injected.
We have used ITP for numerous phosphonate analyses, in cluding (1) determination of N T P electrolysis products I 191, (2) determination of phosphonate thermal stabilities and degradation products, (3) analysis of boiler waters, automobile radiator fluids and detergents for phosphonate content and (4) determination of NTP in dosed rat feeds. ITP is clearly superior for the latter analyses where polyvalent metals extracted from the feed can poison chromatographic columns.
Table 1. Composition of common electrolytes and buffers used for ITP and C Z E
pH 3.3
pH 6.0
pH 8.8
Leading ion Leading counter ion Leader additive Terminating ion Terminating counter ion Terniinatine pH
10 ItlM HCI @- Alanine 0.2 % HPMCh) 10 m M Caproic acid
10 m M HCI L-histidine 0.2 % HPMCh) 10 mM MESCl Trisd) 6.0
10 mM HCI Ammediol”) 0.2 % HPMCbl 10 mM p-Alanine Ba(OH),
11 P caiion electrolytes Leading ion Leading counter ion Terminating ion Terminating counter ion Termin:itine p H
PH 10 mM HCI
ITP anion electrolytes ~~~
C Z t buffers
~
~~~
-
=
-
10 mM Tris
___
pH 4.5 10 mM KOAc HOAc 10 mM HOAc
HCI 8.5
-
pH 7.5
pH 9.0
20 mM Tricinee) 20 mM C H E S 10 mM KCI 10 mM KCI (adjusted to proper pH with 1 M KOH)
a) Ammediol, 2-amino-2-methyl-1,3-propanediol b) HPMC, hydroxypropylmethyl cellulose c) MES, 2-(N-morpholino)-ethane sulfonic acid d) Tris, tris(hydroxymethy1)aminomethane e) Tricine, N-tris(hydroxymethy1)-methylglycine f) CHES. 2-(N-cyclohexylamino)-ethanesulfonic acid
9.0
752
F. S. Stover
Electrophoresis 1990,11, 750-756
+diff.
cond.
I
I
I
14
15
16 min.
: 16 min. 14
ITP will generally dissociate all but the most stable metalligand complexes in the samples [20], particularly using low pH electrolytes. In the above method developments, simple dilution and injection were sufficient to quantitate phosphonates, and no interference was seen when strong acids, glycols, polyacrylates or surfactants were present in the samples.
ct-
1 \
15
Another important phosphonate compound is N-phosphonomethylglycine, or glyphosate, the active ingredient in Roundup@herbicide. ITP separations of this and other herbicides have been described previously I2 1,221. Figure 3 shows our ITP separation of glyphosate and related compounds. Addition of Ca2+ to pH 6 electrolytes improves the separation through metal complexation effects. Analyses for glyphosate have been developed for synthetic samples, plant matter extracts and process streams.
3.1.3 Amino acids and analogs
HCOOH
glyp hosate
1
ITP separations of amino acids suffer from poorer resolutions and sensitivities compared to high performance liquid chromatography (HPLC) methodology. However, nearly complete ITP separation of the 20 common protein amino acids has been obtained [ 231 and no derivatization is necessary. ITP has the advantage of being a rapid and simple method for quantitating a few amino acids and is useful when analyzing complex matrices. ITP may be preferred when other species also must be determined. For example, we have used cationic ITP to simultaneously determine K and lysine 1241. Another application from our laboratory [251 is the analysis of poultry feed for the methionine analog, 2-hydroxy4(methylthio)-butanoic acid (HMB). Figure 4 gives the conductivity record for blank and HMB-containing feeds using pH 6 electrolytes. ITP analysis was found to be faster and more sensitive than G C methods, and was less prone to interferences than HPLC. Total extraction/analysis times of 20 min and sensitivities of0.04 % HMB in feed were obtained.
L. H2OBPCHO
cond.
--r---19
Figure 2. Conductivity and differential conductivity signals from ITP separations of aminoalkylphosphonic acids using p H 6 electrolytes from Table 1 with (a) 0 mM and (b) 2 mM ZnC1, added to leader. Separation current 250 pA for 10 min, detection current 50 pA. Approximately 1 yg each aminoalkylphosphonic acid and0.5 pg phosphate and phosphite iniected. For abbreviations see Section 3.1.2. Reprinted from [ 171, with permission.
21
23 rnin.
Figure 3. Conductivity signal for the ITP separation of glyphosate and related compounds. pH 6 electrolyte from Table 1 with 2 mM CaCI, added to leader. Separation current 200 yA for 15 min, detection current 50 yA. Approximately 0.5 yg phosphite and phosphate and 1 pg other components injected.
+
3.1.4 Polyelectrolytes Polyelectrolytes are often difficult to analyze by HPLC or ion chromatography due to concerns about column poisoning and regeneration. We have found ITP in open PTFE capillaries to be an excellent alternative for polyelectrolyte
Electrophoresis 1990,11, 750-756
Industrial applications of capillary electrophoresis
a
I
--\, A
Figure 4 . ITP conductivity and differential
HMB ‘ONE
conductivity traces for (a) blank poultry feed and (b) feed containing ca. 0.2 96 2-hydroxy-4(methylthio)butanoic acid (HMB) using p H 6 electrolytes from Table 1. Injections (5 pL) 1 gram feed extracted with 10 mL water for 15 min. Reprinted from 1251, with permission.
b-.--;
j cond. t
4
CI-
CI-
-
diff.
L
cond.
CI-
753
A
fumarate
%aleate
C0,‘- + polymer ,methyl
maleate
copolymer and impurities by ITP; (a) standards and (b) 10 pg polymer in p H 8.8 electrolytes from Table 1;(c) 10 pgpolymer
P-ala
analyses. Many polyelectrolytes are mobile in 1TP and determination of both polymers and their impurities can be performed. Figure 5 shows separations obtained with p H 9 electrolytes of a partially butylated styrene-maleic acid copolymer. The polyelectrolyte interferes with determination of the methyl and butyl half esters of maleate using standard pH 9 electrolytes. Advantage can be taken ofdifferential complexation of the polymer with Ca2+(Fig.5c) to achieve separation of half esters, maleate and fumarate. The sloping conductivity trace seen in Fig. 5b is not uncommon in polyelectrolyte separations, and may indicate microheterogeneity of the polymer. In addition to monomer analysis, determination of polyelectrolytes themselves can be performed. ITP has been used in our laboratory to determine po\yacrylates in detergent formulations. Also, we have found the wide pH range available to ITP important for the determination of monomers in polycarboxylates when polymer stability is a concern. Whitlock [26,27] has used ITP to assess the degree of homogeneity, charge density and counter-ion binding in a variety of polyelectrolytes.
species - is seen. Combined UVfconductivity detection was useful for identifying 2-(sulfono)-nonanoylbenzene sulfonate and 2-(sulfono)-nonanoic acid, since standard materials were not available.
3.1.6 Cations One of the most useful applications of ITP is in the determination of cations [30, 3 11. ITP allows simultaneous determina-
t
I
CI-
cond.
7 ” I
3.1.5 Surfactants ITP electrolyte systems have been developed for the analysis of different classes of surfactants 1281. These direct methods offer minimal sample handling compared to extraction and titration methods. Trimethylalkyl ammonium cations with alkyl chain lengths up to 16 are generally mobile in potassium acetate/acetic acid (KOAc/HOAc) electrolytes. Standard p H 6 electrolytes can be used for alkylsulfates and alkylbenzene sulfonates. We have used the methanolic electrolyte systems reported in the literature [29] for the determination of fatty acids in formulated soaps. In some cases, chain length discrimination by ITP is poor. This can be exploited, however, to yield simple class separations since response factors of homologs are similar. Figure 6 shows conductivity and UV signals from the separation of an alkanoyloxybenzene sulfonate synthesis mixture using p H 6 electrolytes. A good separation - based on number of acid groups and size ofeach
I
I
I
I
9
11
13
15 min.
Figure 6 . Separation of 10 pg of a partially purified surfactant synthesis mixture by ITP using p H 6 electrolytes from Table 1. Conductivity signal and UV signal at 254 nm. (1) Sulfuric acid, (2)2-(su1fono)-nonanoic acid, (3) 2-(sulfono)-nonanoyl-p-benzenesulfonic acid, ( 4 ) p-phenol sulfonic
acid, ( 5 ) unknown impurity and (6) nonanoyl-p-benzene sulfonic acid.
754
Electrophoresis 1990,lI, 750-756
F. S. Stove1
'I
Figure 7. Conductivity signals from separations of alkali metals and ammonia in pH 2 cation electrolytes from Table 1 with (a) 0 mM and(b) 5 0 m 18-crown-6 ~ ether added. Reprinted from [241, with permission.
b t i
Rb NH44-K
cond.
1 7 8
10
12 min.
9
13 min.
11
tion of both inorganic and organic bases, often with little method development. We have found many organic bases to be mobile in KOAc/HOAc electrolytes 1321. Cationic ITP applications in our laboratory include quantitating K', Na', NH, +, Ca", trimethyl-, tributyl- and isopropylammonium, guanidine and substituted guanidinium cations, trimethylsulfonium, capryltrimethylammoniuni, para- and diquat, aminotriazole, TRlS, triethanolamine and morpholine in a variety of samples. Neutral ionophores have shown considerable utility for tailoring ITP separations 133, 341. Figure 7 shows a separation developed in our laboratory for alkali metal cations with and without added 18-crown-6 ether 1241. Fifty mM 18-C-6 allows complete separation of alkali metals, while 3 mM 18-C-6 is sufficient for obtaining NH, L/K+separations
3.2 CZE C Z E has shown considerable promise for high resolution analytical 1401 and preparative 1411 separations. C Z E is particularly well-suited to analysis of biological macromolecules 142-441, where theory predicts very high theoretical plate numbers due to small diffusion coefficients. In our laboratories, C Z E has been tested for separations involving peptides and proteins.
a
CI -
b
"'
i
3.1.7 Physical constant determinations C E methods are attractive for the determination of physical constants such as ionic mobilities or single ion conductances, pKa's and metal-ligand binding constants. Hirokawa and Kiso (35-391 have shown the utility of JTP for determining these constants. Advantages of ITP (and CZE) for physical chemistry studies include applicability to niicro samples and on-line purification prior to measurement. ITP was employed when we needed to determine the limiting single ion conductance of glyphosate in order to interpret salt conductivity data. Normal conductance methods were difficult to use with this anion due to its polybasic nature. By employing a pH 4.1 electrolyte system, glyphosate could be buffered predominantly to its monovalent form. ITP runs of glyphosate anion and isopropylammonium (IPA) cation are shown in Fig. 8. Bromate and potassium were used as internal mobility standards for the anion and cation runs, respectively. Using formulas deveioped by Hirokawa and Kiso [351, single ion conductances of glyphosate and IPA were found to be 26.9 and 39.3 S-cm2/eq,respectively. IP A's single ion conductance was checked by independent conductivity measurements and excellent agreement (39.8 S-cmz/eq) was seen.
I I
1 K'
L?
glyphosate
cond.
, 10
11
12
min
13
8
9
10
11
min
Figure 8. Conductivity signals from determinations of limiting single ion conductances (mobilities). (a) Injection of ca. 3 pg each glyphosate and sodium bromate. Electrolytes: leader 10 mM HCI + 5-aminocaproic acid, pH 4.1, leader additive 0.2 % HPMC, terminator 10 mM caproic acid. (b) Injection of 1.5 pg isopropylammonium (IPA) chloride and 1.0 pg KCI in pH 2 cation electrolyte from Table 1.
Electrophoresis 1990,1I, 750-756
155
Industrial applications of capillary electrophoresis
3.2.1 Peptides We have shown that C Z E is capableofhigh resolution separations of biologically important peptides, such as angiotensins I, I1 and I11 [91. In exploring the C Z E behavior of cationic peptides in bare fused silica capillaries, we were interested to see if metallation, as well as protonation, equilibria could be used to enhance separations. Figure 9 shows the C Z E separation of three blocked heptapeptides with one (M), two (D) and three (T) histidines in a p H 7.5 buffer. Without added metal, histidines are mostly deprotonated and the three peptides comigrate. Addition of 1 mM Zn*+allows separation of the peptides through differential metal binding. This separation is comparable to ligand exchange HPLC on immobilized-metal affinity columns. The expected migration order (T > D > M) was not seen in these experiments due to interactions of highly charged Zn-T complexes with the capillary surface. Such interactions will complicate the use of bare silica capillaries for physical constant determinations, and more inert capillaries may be needed. However, Lauer 1451 has given correlations between C Z E mobility and peptide charge and chain length.
separated in a pH 9 buffer. Very high resolutions are obtained as seen by theoretical plate numbers in excess of 100 000. Another important class of proteins are human interleukins. Interleukin-3 is one of a number of colony stimulating factors that aid in bone marrow replication. We have used C Z E to monitor recombinant human interleukin-3 (rh 1L-3)levels in a commercial preparation [471, where human serum albumin is used as a carrier (Fig. 11). Recent work 1481 has shown that nanogram quantities of rh IL-3 can be collected from a C Z E capillary and identified by amino acid sequencing or sodium dodecyl sutfate (SDS)-gel electrophoresis.
I --
0
3.2.2 Proteins C Z E separations involving proteins have been prominent in the literature [46]. Two separations developed in our laboratory are shown in Figs. 10 and 11. Figure 10 shows the separation of two different somatotropins (growth hormones) by CZE. Porcine and bovine somatotropin can be quickly
porcine
bovine
_
5
_7
~
-
~ - *
10
15
-_ min
Figure 10. CZE separation of bovine and porcine somatotropins in pH 9 buffer from Table 1. Protein solutions (4 mg/mL) were injected at 5 kV for 5 s. Separation voltage 15 kV. capillary i.d. 75 pm, length to detector 70cm, overall length 100 cm. UV detection at 230 nm and 0 01 AUFS
a
UV abs.
0
2
4
6
min.
Figure 9. Separation of blocked heptapeptides: (T) Gly-His-Gly-His-GlyHis-Gly, (D) Gly-Gly-His-Gly-His-Gly-Gly and (M) Gly-Gly-Gly-HisGly-Gly-Gly, by CZE using pH 7.5 buffer from Table 1 with (a) 0 mM and (b) 1 mM Zn(CIO,), added. Voltage 15 kV, capillary i.d. 75 pm, length to detector 40 cm, overall length 70 cm. UV detection at 220 nm and 0.01 AUFS. Reprinted from 191, with permission.
I
I
0
5
I
10 min.
Figure 11. CZE separations of (a) recombinant human interleukin-3 (rhIL-3), (b)human serum albumin (HSA)and(c)commercial rhIL-3 containing HSA. Separation conditions the same as Fig. 9 except using pH 9 buffer from Table 1. Reprinted from [471, with permission.
756
F. S. Stover
4 Concluding remarks The applications of C E presented here are intended to highlight the versatility of these separation methods for industrial analysis. From our perspective, the great strength of these techniques lies in their ability to rapidly develop analysis methods. This ability derives not only from flexible operating conditions which can be quickly varied. but also from the hardiness of the separation compartment and minimal sample preparation requirements. In our laboratories, any sample type can be tested under any electrolyte condition without fear of costly equipment replacement or down-time.
An increased emphasis on CZE for small molecule separations is expected with the advent of commercial C E instrumentation using microbore silica capillaries. When developing C Z E separations for small molecules, it would be wise not to overlook the wealth of information on mobilities, buffer systems, complexation phenomena and solvent effects developed over the last 15 years by researchers in ITP. T o date, relatively few C Z E analyses have been developed for small molecule separations. Most of these applications involve use of derivatives [49] or specific detectors [501. The current lack of universal detection, which should take full advantage of the versatility of the technique, limits applications. One approach has been the use ofindirect detection [ 5 1-531, but this method is not without limitations regarding sensitivity and dynamic range. An appealing alternative in sub-100 pm C E separations is operation in the ITP mode. Here, lack of universal detection is not as great aproblem, since the detector can serve qualitatively to mark zone boundaries. ITP separations with UV-absorbing impurities as markers already have been shown using fused silica instrumentation [ 541. The indirect-UV method of Reijenga et al. [551 for ITP also should be useful for universal detection in modern instruments. Finally, greatly increased sensitivities and load capacities may be possible through capillary coupling [561 to microbore fused silica. These factors indicate that the ITP mode may remain the method of choice for developing small molecule separations. Received December 14, 1989
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