516 A. Cifuentes and H. Poppe Electrophoresis 1995, 16, 516-524

Alejandro Cifuentes Hans Poppe

Effect of pH and ionic strength of running buffer on peptide behavior in capillary electrophoresis: Theoretical

~abomtory of halytical Chemistry, calculation and experimental evaluation University of Amsterdam

The effect of pH and ionic strength of running buffer on peptide behavior in capillary electrophoresis (CE) is studied. A system for predictions of peptide migration in CE (SPPMCE) developed in our laboratory has been tested in a wide range of pH and buffer concentrations. The SPPMCE consists of a com- puter program for calculating peptide pK, values, an equation which relates peptide structures to their electrophoretic mobilities and a coupled computer program for the prediction of electropherograms. More than 25 different buf- fers have been employed, covering a pH range of 2-11 and a concentration range of 5-100 mM. Results from experiments are compared with the theore- tical predictions. Good agreement is observed, which confirms the utility of the SPPMCE and allows fast and easy optimization of peptide separations in CE, with nothing more than the amino acid sequence of the linear peptide as the input.

1 Introduction

In recent years the separation of peptides and proteins by capillary electrophoresis (CE) has become one of the most exciting application fields of this technique [ 1-41. Analytical and sequence data as well as information about purity and structural changes of peptides and pro- teins can be obtained from CE analysis of these biopo- lymers [2, 5-71. The separation mechanism in CE is based on the differing electrophoretic mobilities of the analytes in an electric field due to differences in the charge-to-size ratio of the solutes. Considering the amphoteric character of peptides, the pH of the running buffer appears to be one of the key parameters to be stu- died for obtaining a good separation [8-161. Also, the buffer concentration can be manipulated and can have a beneficial effect on the separation performance [6, 10, 12, 13, 151. The procedure generally followed for obtaining a good separation of peptides is to make a more or less systematic study about these parameters, preparing and running several buffers with different concentrations and pH until reaching the conditions in which peptides are conveniently separated [8, 10, 11, 151. However, this procedure is always a time-consuming task.

Several computer programs have been developed to predict the electrophoretic behavior of small molecules and to obtain optimum separations in CE [16-271. These programs reduce the long time normally needed to improve the quality, in terms of peak shape and resolu- tion of CE separations, and at the same time facilitate the identification of solutes. However, the possibilities to employ these programs decrease the higher the analyte complexity in terms of number of charged groups [21,

Correspondence: Dr. Hans Poppe, Laboratory of Analytical Chemistry, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands (Tel: 31-20-525-6515; Fax: 31-20-525- 6638)

Abbreviations: SPPMCE, system for prediction of peptide migration in CE; TRICINE, N-tris[hydroxymethyl]methyl-glycine

Keywords: Capillary electrophoresis / Computer simulation / Peptide analysis

281 (e.g., peptides and proteins). The main difficulty in this case comes from the few data available in literature about the required input parameters (pK, and p values) for these biopolymers [29-311.

In a previous paper [32] we showed the possibilities of a System for Predictions of Peptide Migration in CE (SPPMCE) developed in our laboratory. This system consists, briefly, of a computer program for calculating peptide pK, values, an equation which relates the pep- tide structure to its electrophoretic mobility and a cou- pled program for prediction of electropherograms. The objective of the present investigation was to test the validity of the SPPMCE in a wide range of pH and buffer concentrations. More than 25 buffer solutions were employed, covering a pH range of 2-11 and a con- centration range of 5-100 mM. Results from experi- ments and from predictions are compared.

2 Materials and methods

Separations were carried out using a Prince (Lauer-Labs, Emmen, Netherlands) injection system with temperature controller, connected to a Linear M-200 variable-wave- length UV-Vis detector (Linear Ins. Corp., Reno, NV, USA) operated at 210 nm. A fused silica capillary (Poly- micro Technologies Inc., Phoenix, Arizona, USA) with 75 pm I D and 360 pm OD was used; the total length of the capillary was 63 cm and the effective length (from the injection point to the detector) was 50 cm. The injec- tion was carried out at the anodic side using controlled pressure for a determined time. The computer programs employed were: (i) one written at our laboratory for the simulation of the electrophoretic separations (whose characteristics are described in [16]) and (ii) a custom program to carry out the pK, and mobility calculations [32]. Both programs were used on a 486DX-33 micro- processor-based PC. Peptides GGG, AGG and LGF were purchased from Nutritional Biochemicals Corpora- tion (Cleveland, OH, USA). Peptides GGP and LGG were from Fluka (Fluka AG, Buchs, Switzerland). All the long peptides were purchased from Bachem Feinchemi- kalien AG (Bubendorf, Switzerland) and used as

0 VCH Verlagsgesellschaft mbH, 69451 Weinheim, 1995 0173-0835/95/0404-0516 $5.00+.25/0

Electrophoresis 1995, 16, 516-524 Simulation of peptide behavior in capillary electrophoresis 517

received. The peptides were dissolved in water, previ- ously purified by passage through a PSC filter assembly (Barnstead, Boston, MA, USA), at the concentrations indicated in each case. The samples were stored at -20°C and heated to room temperature before use. Malic acid (J. T. Baker Chemicals, Deventer, Netherlands), succinic acid (BDH Laboratory Chemicals Division, Poole, England), malonic acid, TRICINE, MES and CAPS (all from Aldrich, Axel, Netherlands), were used in the dif- ferent running buffers (see Table 1). The buffers were stored at 4°C and heated to room temperature before use.

Table 1. Concentrations, pH, pK, values and electrophoretic mobilities (v),) of the 27 buffer components employed in this work

Compound Concentration Buffer PKI pK2 1.11. p2. (mol/L) PH

Malonic acid 0.05 2.25 2.847 5.696 -40.7 -67.0 2.50 2.75 3.0

3.15

4.25 4.5 5.0 5.5 6.0 6.5

Malic acid 0.05 3.25 3.469 5.050 -32.6 -59.0

Succinic acid 0.05 4.0 4.207 5.638 -33.0 -60.9

MES 0.05 7.0 6.10 -28.0 TRICINE 0.05 8.0 8.14 -21.1

8.5 9.0

9.75 10.0 10.5

0.005 10.0 0.01 10.0 0.02 10.0 0.03 10.0 0.05 10.0 0.1 10.0

CAPS 0.05 9.5 10.4 -18.2

a) All electrophoretic mobilities are given for the separate ion species i.e. ions with a particular number of protons attached, and in m2/sV units.

FROM EWERIMWTS OR FREELY CHOSEN

3 Results and discussion

In CE, efficiency, selectivity, peak shape and with them resolutions in the electropherogram are strongly influenced by the pH [8, 13-15] and the type and con- centration of the buffer [6, 151. Therefore, the optimiza- tion of a peptide separation involves a laborious task which normally starts with preparing a set of buffer solu- tions with several pH values and buffer concentrations. The goal is to fully separate and identify the peptides. The latter can be done by matching peak position when standards are available, or, if they are not, by techniques such as RP-HPLC and FAB-MS [lo, 11, 141. In order to check the validity of a tentative assignment in an early stage, a method that could predict the peptide behavior in CE directly from their structure would be helpful. As we showed in a previous paper [16] predictions of separa- tions in CE can be achieved if the necessary input data are obtained. A general scheme of the SPPMCE require- ments is shown in Fig. 1: to obtain the complete electro- pherogram it is necessary to introduce the following data into the computer program: capillary dimensions, injec- tion time and voltage (or injected plug length), separa- tion voltage, electroosmotic flow and sample concentra- tion. These parameters can be found easily, when a given experiment is simulated or freely chosen, by testing dif- ferent separation conditions. The parameters for the run- ning buffer (pK, and electrophoretic mobilities) are usu- ally easily obtained from the literature [33-351.

The main complication comes when, e.g., peptides are the sample, since only very few peptide pK, and mobility values are available in the literature [29,30]. To calculate the pK, values of each peptide we have written a com- puter program which allows us to find these pK, values based on the structure of peptides [32]. For estimating the electrophoretic mobility of the different peptides we have used the equation [32]

1758 Log(1+0.297q) c L = ~0 411

PEPTIDE STRUCTURE n pK VALUES \ p VALUES 1

' 2 I

t -

where p is the electrophoretic mobility given in lo9 mZ/sV, q is the value of species' charge and M its molec-

J

Figure 1. Scheme and requirements of SPPMCE.

518 A. Cifuentes and H. Poppe Electrophoresis 1995, 16, 516-524

Table 2. pK, values and electrophoretic mobilities as obtained from the procedure of [32] and used for the predicted electropherograms of the five peptides in Figs. 3 and 7’’

No. Peptide Mobility and pK, values for the different peptides

pK 11.21 1.77 4.16 2.91

pK 8.58 5.45 4.23 3.24

pK 12.4 10.18 6.68 5.27 2.78

pK 13.21 10.3 7.36 6.3 3.74 2.61

pK 8.52 5.18 4.22 3.31

1 LQAAPALDKL p -20.49 -11.43 0 11.43 20.49

2 WAGGDASGE c~ -30.43 -22.27 -12.42 0 12.42

3 FHPKRPWIL p -10.80 0 10.80 19.36 26.45 32.51

4 SYSMEHPRWG 1.1 -25.96 -19.0 -10.6 0 10.6 19.0 25.96

5 ELAGAPPEPA IJ. -29.4 -21.25 -11.85 0 11.85

a) All electrophoretic mobilities are given for the separate ion species, i.e. ions with a particular number of protons attached, and in lom9 m2/sV units.

ular mass. The program in which these elements have been integrated allows the prediction of the electrophe- rogram with just the amino acid sequence of the peptide as the input. Thus far it works only for linear, non- branched, noncycled peptides. The number of acid-base active groups can be as large as 14.

3.1 Peptide behavior depending on buffer pH

In order to study the dependence of peptide behavior on the pH of the running buffer we have used more than 20 different buffer solutions covering a pH range of 2-11. The characteristics of these buffers and their pK, and the mobility values given for the separate ion species are shown in Table 1: As a general rule all the buffers were employed in a pH range of k 1 unit around the pK, of the respective acid and in a concentration of 0.05 M.

The group of peptides used as sample in this part of the work and their respective pK, and mobilities are shown in Table 2. The peptides used range from highly basic (e.g. peptide 3) to highly acidic (e.g. peptide 2). In Figs. 2 and 3, we show a comparison between experimental sep- arations (Fig. 2) and their respective simulated electro- pherograms (Fig. 3) at different pH values. As can be seen, good agreement was obtained for these separa- tions, with a correct prediction of the change of migra- tion order for peaks 2 and 5 in a difference of pH of 0.25 units between each separation.

Good agreement was also observed for peptide 3 since, in all the pH ranges studied, this peptide always ap- peared in the first position, both in the real separations as well as in their respective simulations. As can be easily deduced looking at the set of pK, values calculated for this analyte (Table 2), in the range of pH used (2-10.5) this peptide should take the highest positive charge compared to the other peptides. At these condi- tions, the apparent electrophoretic mobility (electroos- motic flow plus effective electrophoretic mobility) of peptide 3 should be always larger than that of the other peptides, as was corroborated by the real electrophero- grams. In spite of this good agreement, the peak shape sometimes could not be well predicted for this peptide. We assume that this analyte could be highly adsorbed on the capillary wall and this effect has not been included in the program for simulating electropherograms. How-

A

B

I I I I I I I 24 20 16 12 8 4 0

Figure 2. Influence of the running buffer pH on the migration order of the peptides from Table 2. Sample concentration: 1) 1.68 X M, 2) 1.69 X M, 3) 1.0 X M, 4) 0.50 X M and 5) 2.10 X M. Conditions: capillary length, 63 cm (SO cm to detector); 75 pm ID, 360 pm OD. Run voltage, 18 kV. Injection: 10 mb for 0.10 min. Buffer: sodium malonate, 0.05 M. (A) pH 2.25, (B) pH 2.50 and (C) pH 2.75. UV detection at 210 nm.

Time (min)

Electrophoresis 1995, 16, 516-524

0 1

0

Simulation of peptide behavior in capillary electrophoresis 519

- 0

0 0

0 a

A

B

21 I s C

Time (min) Figure 3. Simulation of electropherograms from Fig. 2. All conditions as in Fig. 2.

ever, knowing the approximated set of pK, values directly from the sequence of peptides as given by the SPPMCE, and taking into account that the larger the basic character of a biopolymer, i.e., high number of posi- tive charges, the larger the adsorption expected on the capillary wall [36, 371, the SPPMCE can also be an important source of information about the expected occurrence of adsorption effects prior to obtaining the real electropherogram.

Since in almost all the separations the two pairs of pep- tides 4-1 and 5-2 appeared very close, and in order to compare experiments and predictions in all the ranges of pH studied, we have employed the parameters

a41 = (p4-p1)/G4,1 (2)

and

= (p5-p2)/&,2 (3)

where p, , p2, p,, p, are the mobilities of peptides 1, 2, 4, and 5, respectively, and and Li,,2 the average mobili- ties of peptides 1,4 and peptides 5,2. Thus, a change in the sign of a,, or as, from positive to negative or vice versa, means a change in the separation order between these peptides.

In Figs. 4 and 5 we show the values of a,, and a,,, ob- tained from experiments and simulations, as functioning on the buffer pH. As can be seen in Fig. 4, good agree- ment was obtained for peptides 1 and 4 (a41 value) in all the range of pH studied although small differences were

01 I I I I i I I I I I I 1 2 3 4 5 6 7 8 9 10 11

PH Expenmental Predicitlon

Figure 4. Comparison between predicted and experimental analysis, e.g. value, for peptides 1 and 4 of Table 2 in all ranges of pH stu- died. The buffers employed are given in Table 1. All conditions as in Fig. 2.

a *= p5-p21@5+p2)/2 02

PEmDE 2 AND 5

0.2 1 I I I ,D'.:::;.,;l I , , , I I I , , , , I

. , . , . . . . : .

-0.3

0.4 I 1.5 z 2.5 3 3.5 4 4.5 5 5.5 e 8.5 7 7.5 e 8.5 Q 9.5 10 10.5

PH Experimental Prediction

0 ... . ..... . . . ...

Figure 5. Comparison between predicted and experimental analysis, e.g. aS2 value, for peptides 2 and 5 of Table 2 in all ranges of pH stu- died. The buffers employed are given in Table 1. All conditions as in Fig. 2.

observed in the quantitative value of a41. These small dif- ferences can be linked to the accuracy of the data neces- sary for the simulations, namely mobilities and pK, values of the peptides. This phenomenon has already been observed and described [38], and, in our particular case, it could be related to different effects whose influence has not been included in the calculation of pK, and mobility values of peptides (e.g. ionic strength, hydrophobicity [7], different orientation of the side chains [7], etc.) and which could also explain the differ- ence in sign observed in Fig. 5 between the predicted and experimental a,, value in the pH range of 5-6.5.

As an application of the SPPMCE we have simulated in Fig. 6 the separation of 14 different motilin peptides and the result has been compared with the real separation taken from the work of Florance et al. [14]. The data set (pK, and mobilities) employed in the simulations of these 14 peptide and their respective sequence of amino acids are given in Table 3. As can be seen in Fig. 6 the agreement obtained between simulation (Fig. 6b) and experimental separation (Fig. 6a) is good. A difference was observed for peptide 3 between both results. In their work, Florance et al. [14] also observed an unexpected

520 A. Cifuentes and H. Poppe Electrophoresis 1995, 16, 516-524

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Table 3. pK, values and mobility values as in Table 2 used for the predicted electropherograms of the 14 motilin peptides in Fig. 6a)

No. Peptide Mobility and pK, values for the different peptides EKERNKGQ I-I -28.59 -20.92 -11.67 0 11.67 20.92 28.59 35.13

pK 11.72 10.82 9.45 7.42 3.95 3.09 2.23

pK 11.73 10.84 9.70 8.81 6.95 3.81 2.95 2.10

pK 10.81 9.76 7.37 4.35 3.32

pK 12.29 11.47 10.69 9.93 9.19 8.29 4.34 3.58 2.89 2.11

pK 12.29 11.46 10.68 9.90 9.09 7.12 4.36 3.60 2.91 2.13

pK 12.16 11.10 10.32 9.50 7.35 4.75 3.95 3.21 2.36

pK 11.89 10.73 9.71 7.36 4.67 3.77 2.81

pK 10.91 9.93 7.39 4.46 3.39

pK 10.82 9.82 8.64 4.32 3.29

pK 10.81 9.77 7.76 4.34 3.31

pK 11.10 10.08 7.61 5.12 4.13 3.09

pK 7.54 3.65

pK 10.64 7.46 3.74

pK 10.84 7.72 5.02 3.54

LQRLQEKERNKGQ ~1 -23.29 -17.04 -9.51 0 9.51 17.04 23.39 28.62 33.27

FVPIFTYGELQRLQ I-I -22.81 -16.69 -9.31 0 9.31 16.69

PIFTYGELQRLQEKERNKGQ -28.19 -24.25 -19.73 -14.44 -8.05 0 8.05 14.44 19.73 24.25 28.19

FVPIFTYGELQRLQEKERNKGQ I-I -27.09 -23.30 -18.96 -13.88 -7.74 0 7.74 13.88 18.96 23.30 27.09

FVPIFTYGELQRLQEKERN -28.51 -24.53 -19.96 -14.60 -8.15 0 8.15 14.60 19.96 24.53

FVPIFTYGELQRLQEK I-I -26.46 -21.53 -15.76 -8.79 0 8.79 15.76 21.53

FVPIFTYGELQR -24.27 -17.76 -9.91 0 9.91 17.76

PI FTYG E LQRLQ P -24.31 -17.79 -9.92 0 9.92 17.79

VPIFTYGELQRLQ I-I -23.66 -17.32 -9.66 0 9.66 17.32

FVPIFTYGELQRLQE CI -27.20 -22.13 -16.20 -9.03 0 9.03 16.20

FVPIF I.I -14.11 0 14.11

FVPIFTY I-I -21.87 -12.20 0 12.20

FVPIFTYGE IJ. -27.63 -20.22 -11.28 0 11.28

a) All electrophoretic mobilities are given for the separate ion species and in mZ/sV units.

behavior for this peptide when they plotted log q/Mt3 against the migration time for these 14 motilin peptides. The influence of the secondary structure and hydropho- bicity of this peptide were given as possible explanations for this unexpected behavior. Unfortunately, at the pre- sent state of the SPPMCE, there is no way of knowing in advance that a peptide will not match the theoretical prediction, since, as was already discussed above, several parameters which can affect the pK, and mobility values of the peptide have not been included.

3.2 Peptide behavior depending on ionic strength of buffer

The influence of the ionic strength of the running buffer on the peptide behavior in CE has been related to dif- ferent phenomena. First, the variation of the ionic strength of the separation buffer is directly related to the variation of the ionic double layer thickness near the capillary wall and around the peptide, which generate a modification of their zeta potentials and, as a result, a variation of the global electrophoretic mobility of pep- tides. In general this variation affects the migration times and resolution of the separation [6, 151. Second, the relation between the buffer and sample concentra- tions can originate variations on the peak shape, since employing similar concentrations for both solutions can result in overloading phenomena, leading to peak defor- mation [16]. Third, the use of buffer solutions with high ionic strength can produce undesired effects related to the generation of heat. If the heat is not properly dissi- pated by the capillary, the viscosity of the buffer and the electrophoretic mobility of the analytes can be modified, bringing about variations in the efficiency and resolution of the separations [6, 15, 391. In an extreme case, the

high temperatures inside the capillary can produce the denaturation of the biopolymers [40].

In order to test these effects, we employed six buffer solutions at pH 10 and different ionic strengths covering the range of 5-100 mM (see Table 1). As analytes, we employed the set of peptides shown in Table 4 and pep- tides 1, 2, 4 and 5 listed in Table 2. Also, several separa- tions of peptides found in the literature [13] which show the effect of the ionic strength of the buffer on the sepa- ration were simulated. In order to check whether the undesired heat generation was unimportant, we plotted, for the same separation voltage, the electric current against the buffer concentration. In all cases, straight lines with correlation coefficients higher than 0.9990 were obtained. Also, the power dissipation was calcu- lated and the values were never higher than 0.8 W/m. From these results, we concluded that no significant heating effect was present.

Table 4. pKa values and electrophoretic mobilities as in Table 2 used for the simulated electropherograms of the five peptides in Fig. 11')

No. Peptide Mobility and pK, values for the different peptides

1 LGF fl -18.20 0 18.20

2 G G P ~1 -21.27 0 21.27

3 G G G I-I -23.02 0 23.02

4 LGG p -20.69 0 20.69

5 AGG -22.35 0 22.35

pK 8.06 3.47

pK 8.27 3.21

pK 8.27 3.30

pK 8.06 3.30

pK 8.24 3.30 a) All electrophoretic mobilities are given for the separate ion species

and in mZ/sV units.

Electrophoresis 1995, 16, 516-524 Simulation of peptide behavior in capillary electrophoresis 52 1

0

I 0 MINUTES 15

12 l4

B 9 8.10 11

I I

0.0 4 8 T

13

2 1 A

B

C

'1 2 I I I I 1

Time (min) Figure 6. Electropherogram of 14 motilin peptides (see Table 3). Sample concentration: M for each peptide. (A) Conditions: capil- lary length, 72 cm (48 cm to detector); 50 wm ID, 360 wm OD; separa- tion voltage, 30 kV; buffer, 0.02 M sodium citrate, pH 2.5; UV detec- tion at 200 nm. Redrawn from [14]. (B) Simulated electropherogram: electroosmotic flow was considered equal to we0 = 14 X m2/sV.

In Figs. 7 and 8 three real separations at different ionic strength and their respective simulations are shown. As can be seen, the peak height as well as the efficiency and resolution of these separations increase with the ionic strength. For peaks 5 and 2 in Fig. 7a, a variation is ob- served in their peak shapes, passing from frontal peaks (Fig. 7a) at 0.005 mol/L to tailing peaks (Fig. 7b) at 0.02 mol/L. The simulated electropherograms agree quite well with all these effects as can be seen in Fig. 8 (note that in Fig. 8a, b and c the y-scale is different).

In Fig. 9 the experimental and predicted resolution between peaks 5 and 2 against the ionic strength is

Figure Z Influence of the ionic strength of the buffer on peak shape of peptides from Table 2. Buffer: CAPS, at pH 10. (A) 0.005 M, (B) 0.02 M and (C) 0.1 M; pH 10. All other conditions as in Fig. 2.

shown. Resolution was calculated from electrophero- grams using the equation

(4)

where tz and t, are the analysis times for peptides 2 and 5, respectively, and wz and w, their respective baseline peak widths. The agreement between both results is good, indicating the same trend although a small quanti- tative difference is observed. The explanation given above ( i e . , the ionic strength of the buffer, as well as hydrophobicity [7] and different orientation of the side chains [7] of peptides not included in the calculation of pK, and mobility values) could explain the differences observed in this case.

522 A. Cifuentes and H. Poppe

2 = 6.OE-5

I ' 4 ' 4.OE-

2.OE-5

I I 9.0

A 1 .OE-

I

Electrophoresis 1995, 16, 516-524

A

A

1 .BE-4

8.OE-5 I 1.2E-

B

7.

C

1.2E

B

C

I I I I I 7 6 5 4 3

-rim M)

Figure 10. Influence of ionic strength of the buffer on peak shape of peptides from Table 4. Buffer: CAPS, pH 10. (A) 0.005 M, (B) 0.02 M

to 2 X 10.' M. All other conditions as in Fig. 2.

Time (min.) Figure 8. Respective simulated electropherograms of Fig. 7, All condi- and O'' M; pH lo' concentration ranging from

tions as in Fig. 7.

Resolution

0 0.02 0.04 0.06 0.08 0.1

Concentration (molll) Exprimsntal Prediction _____ ___._

Figure 9. Experimental and predicted resolution for peptides 5 and 2 from Table 2 against buffer concentration. Conditions as in Fig. 7.

In Figs. 10 and 11 the real and simulated electrophero- grams of the peptides in Table 4 are' shown as a function of the varying ionic strength of the running buffer. Although the correct peak order was predicted as well as the increase in efficiency and peak height, some differ- ences can be seen between the simulations and real sep- arations in the peak shape and resolution of peptides 2, 3 and 4.

In Fig. 12 the separation of three very similar 8-endor- phin fragment peptides depending on the ionic strength of the buffer is shown. The real separations (Figs. 12a, b and c) taken from [13], are compared to their respective simulated electropherograms (Fig. 12d, e and f). As can be seen, the migration order as well as the improvement of resolution were correctly predicted, showing good agreement between both results.

Electrophoresis 1995, 16, 516-524

0.0016 2

5 4 B 0.0012 I 3

-.

0.0008

0 0004 1

1. - h 0.0

Simulation of peptide behavior in capillary electrophoresis 523

5 0.w1s

0.0012

I O.ooO8

O.ooc4

C _.

I , ( 2

_.

1 n I , 4 1 , 0.0

_.

0.oOW o,mj A

0.w20 i

0.w20 -I

Time (min)

Figure 11. Respective simulated electropherograms of Fig. 10. All con- ditions as in Fig. 10.

0 5 10

4 t

8

4 Concluding remarks

SPPMCE has been tested in a wide range of pH and buffer concentrations. Results from experiments have been compared with those obtained from SPPMCE. In general, good agreement was observed between both results, which confirms the utility of the SPPMCE as a helpful tool for identification of peptides. This allows its use to obtain fast and easy optimization of peptide sepa- rations in CE. Nevertheless, many improvements are still necessary. For instance, the inclusion of the ionic strength effect into the program for pK, calculations could give more accurate values, as well as charge and electrophoretic mobilities, which should improve the agreement between real electropherograms and predic- tions. The possibility to work with double peptide chains and non-common amino acids, i.e. hydroxyproline, car- boxyglutamine, ornithine, etc., as well as the effect of other parameters such as hydrophobicity and secondary structure, should also be included in order to make pre- dictions in a wider field of applications.

This work was supported by the Commission of the Euro- pean Communities (Human Capital and Mobilily Pro- gramme, bursary # ERB4001 GT920989).

Received September 20, 1994

0 5 10 16

D

1 1

Figure 12. CE separation of 8-endorphin fragment peptide depending on ionic strength of the buffer solution. Condi- tions: capillary length, 57 cm (50 cm effective length); 75 ym ID. Buffer: sodium phosphate, pH 2.20. (A) 25 m ~ , run voltage 25 kV; (B) 50 mM, run voltage 20 kV; (C) 100 mM, run voltage 20 kV. Sample concentration: 0.2 mg/mL of each peptide. Injection: 25 nL. Detec- tion: 214 nm. Redrawn from [13]. (D),

8 2 8 12 16 (E) and (F) simulated electropherograms (note that the time scale in x-axis is dif- ferent); conditions as in (A), (B) and (C),

~~ Time (min) respectively.

524 A. Cifuentes and H. Poppe Electrophoresis 1995, 16, 516-524

5 References

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[3] Grossman, P. D., Colburn, J. C., Lauer, H. H., Nielsen, R. G. , Riggin, R. M., Sittampalam, G. S., Rickard, E. C., Anal. Chem.

[4] Cobb, K. A., Novotny, M., Anal. Chem. 1989, 61, 2226-2231. [5] Nielsen, R. G., Riggin, R. M., Rickard, E. C. , J. Chromatogr. 1989,

[6] Rickard, E. C., Strohl, M. M., Nielsen, R. G., Anal. Biochem. 1991,

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