ANALYTICAL

BIOCHEMISTRY

94, 60-64 (1979)

Quantitative Determination of Amino Groups on Derivatized Controlled Pore Glass: A Comparison of Methods C. C. Y. LEE AND G. MARC LOUDON Department of Medicinal

Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907 Received September 21, 1978

The following methods of determining covalently bound ammo groups were tested for self-consistency and convenience on aliquots of the same sample of controlled pore glass which has been derivatized with triethoxyaminopropylsilane (aminopropyl glass): (i) the determination of bound picrate; (ii) the determination of 2-hydroxy-l-naphthaldehyde after Schiff base formation on the aminopropyl glass; (iii) direct nonaqueous titration of the bound amines with HCIO, in acetic acid and with CF,SO,H in acetic acid; and (iv) titration by Hg(NO& solution of chloride bound by treatment of the aminopropyl glass with HCl. All methods gave the same results within the precision of measurement. Methods (i) and (iv) appear to be the most convenient.

When carrying out chemistry on solid supports, one frequently has to rely on chemical methods for the quantitative determination of functional groups immobilized on these supports. In the case of solid phase peptide synthesis (1,2), the concentration of amino groups present on the divinylbenzene-crosslinked polystyrene support (Met-Afield resin) becomes of crucial importance (3-9). Recently, we have found that porous glass is a useful insoluble matrix for the degradation of peptides from the carboxyl terminus (10,ll). Likewise, this material also appears to be a suitable insoluble support for the Edman degradation of peptides (12). In view of the increasing importance of porous glass in peptide sequencing and liquid chromatographic ( 13,14) applications, the determination of functional groups present on these materials assumes special relevance. Faced with the problem of determining covalently bound amino groups on glass supports, we have been led to compare several amino group determination methods which have in various forms been used for a similar purpose on Merrifield resins and examine these various methods for self0003-2697/79/050060-05$02.00/O Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.

consistency and relative convenience. This paper reports the results of this comparison. MATERIALS

AND METHODS

Controlled pore glass (CPG-lo), Electronucleonics, Inc., 120/200 mesh, mean pore diameter 253& was first stirred in concentrated nitric acid at 85-90°C for 4 h (15) and then washed thoroughly with 2 liters of deionized distilled water before it was refluxed in water for another 2 h. The nitric acid treatment is crucial if high backgrounds are to be avoided in the tests below. The glass was dried in V~CUO and then allowed to react with triethoxyaminopropylsilane (10,ll). We have never carried out these procedures without the initial drying/degassing step included; therefore, the consequences of omitting either of these steps have never been systematically examined. The batch used for all of the tests reported here was found to have an amino group content of about 77 pmol/g. On different occasions, amino group incorporations of up to 130 ,umol/g have been observed with different batches of glass. No effort was made 60

QUANTITATIVE

DETERMINATION

to maximize amino group incorporation. The derivatized glass (aminopropyl glass) was again cleaned and dried before being deprotonated with 6 bed vol of 5% triethylamine in methylene chloride followed by 2 bed vol of dried methanol (6). All tests below were carried out on this batch of glass within 48 h after preparation. All reagents used are analytical grade unless specified otherwise. Picrate test (6,8). Aminopropyl glass (66.7 mg) was washed twice with 2 ml of picric acid solution (0.1 M in methylene chloride) followed by washing with 40 ml of pure methylene chloride. Pure NJ-diisopropylethylamine (distilled from CaH,) was added to the glass (two 2-ml portions intervened by 2 ml of methanol). The collected washings were diluted to 250 ml with 95% ethanol. The extinction coefficient of the amine-picrate complex was determined to be 14,500 at 358 nm (6) from a Beer’s law plot. Aldehyde test (3,9). Aminopropyl glass (36 mg) was immersed in 5 ml of 0.2 M 2hydroxy-1-naphthaldehyde in dimethylformamide (DMF)’ for 14 h. First washed with 20 ml of methylene chloride followed by DMF (two 5-ml portions), it was subsequently flushed with 40 ml of 95% ethanol. Benzylamine in ethanol (10 ml, 0.4 M) was added to the glass and the solution was allowed to stand for another hour. It was then generously washed with ethanol until the glass became white again. The washings were collected, diluted to 250 ml, and the absorption measured at 420 nm. The extinction coefficient of the Schiff base formed by benzylamine and the aldehyde was determined to be 10,900 at 420 nm from a Beer’s law plot (9). Nonaqueous potentiometric titration (4,I6). Anhydrous. HCIOl, 0.0514 M in acetic acid (HOAc), was formed by carefully and slowly adding acetic anhydride to ’ Abbreviations used: DMF, HOAc, acetic acid.

dimethylformamide;

OF AMINO

GROUPS

61

a solution of 70-72% perchloric acid in acetic acid (17). The titrant was standardized against potassium acid phthalate in acetic acid with methyl violet indicator (16). Although the use of HClOJacetic acid has been indicated to be routine, and several authors (17,18) even indicate a preference for anhydrous HClOJHOAc over other less hazardous titrants, the hazards of anhydrous HC104 are well known, and safety precautions (19) drawn up following one serious accident involving a large amount of this material specifically suggest avoiding anhydrous HClO, in any form. Although we used small volumes of this titrant and encountered no problems with it, we suggest that since FS03H and CF,S03H are now readily available, and these materials have acid strengths comparable to those of perchloric acid, use of these materials should replace the use of anhydrous HClO, (see below). Into a 30-ml beaker were placed 958 mg of aminopropyl glass and 20 ml of glacial acetic acid. This suspension was titrated against 0.0514 M perchloric acid in glacial acetic acid. Each addition of titrant was followed by bubbling of nitrogen for 3 min into the solution gently through a disposable pipet to agitate the solution without crushing the glass. A Radiometer pH meter with a combination electrode (glass vs calomel) was used with the meter on the millivolt scale. Anhydrous trifluoromethanesulfonic acid was dissolved in acetic acid and was standardized against potassium acid phthalate (16,18). Aminopropyl glass (1 .O g) was titrated against the CF,SO,H/HOAc titrant, and a confirming picrate test was carried out on this sample. Chloride titration (5,20,22). Aminopropyl glass (66.7 mg) was washed with 20 ml of 1 M HCl followed by 40 ml of deionized distilled water and then 5 ml of methanol. The glass was then successively washed with 4 ml of 10% triethylamine in DMF, 2 ml of DMF, and 4 ml of methanol. The three washings were collected and titrated with mer-

62

LEE TABLE

AND

LOUDON

1

TABLE

THE DETERMINATION OF AMINO GROUPS ONAMINOPROPYL GLASS BY THE PICRATE TESTY Determination

Absorption at 358 nm Amine incorporation (P”mc) Average

amino group

THE DETERMINATION OF AMINO GROUPS ON AMINOPROPYLGLASS BY THEALDEHYDETEST

No.

Determination

1

2

3

4

0.311

0.2%

0.287

0.292

80.4 incorporation:

76.5

74.2

75.5

76.7 f 2.7b ymoUg

1 Weight of glass (mg) Absorption at 420 nm Amine incorporation

(pmollg) Average

a Aminopropyl glass (66.7 mg) was used for each determination. b Standard deviation.

curie nitrate solution which had been standardized against KCl. The chloride titrations were carried out according to the following procedure. To the collected washings (or to a standard chloride solution) 20 ml of water and 80 ml of ethanol were added. Bromophenol blue (5 drops of a 1% solution) was added to the washings and the color changed to blue. Nitric acid (0.5 M) was added dropwise to the solution until the color turned yellow. Finally, 5 drops of 1% diphenylcarbazone indicator were added and the titration with Hg(NO& commenced. The endpoint was indicated by a sudden color change to orchid-pink, indicating mercuric ion present in excess over that needed to complex chloride ion. RESULTS

2

AND DISCUSSION

Controlled pore glass was prepared and derivatized with triethoxyaminopropylsilane as indicated in the previous section to yield a glass containing covalently bound aminopropyl groups (aminopropyl glass). The various amine incorporation tests were carried out for comparison on various samples of the same batch of aminopropyl glass. The results of the picrate test, aldehyde test, and chloride titration are presented, respectively, in Tables 1, 2, and 3. The nonaqueous titration results are presented in Fig. 1 (for titration with HCIOI in acetic acid) and Fig. 2 (for titration with

2

3

4

36.00 0.121

46.34 0.157

36.00 0.130

49.73 0.168

77. I

78.3

82.8

77.1

amino group Incorporation:

u Standard

No.

78.7 f 2.7” pmollg

deviation.

trifluoromethanesulfonic acid in acetic acid). In Table 4, the results of all the tests are compared with respect to amine incorporation, amount of glass required, and time required. We note that the level of precision in the various tests is considerably below that observed in similar tests employed on the Merrifield resin. At least two factors are responsible for this observation. One is the lower level of amine incorporation per gram of support. It is recognized (9) that ligand incorporation levels obtainable on glass supports are considerably-perhaps as much as an order of magnitude-below those possible on Merrifield resins. Furthermore, the amine incorporation in the present study was not maximized. Second, except for the nonaqueous titration experiments, conTABLE

3

THE DETERMINATION OF AMINO GROUPSON AMINOPROPYLGLASS BYTHE CHLORIDE~ONTITRATION" Determmation

Amount of titmntb Amine incorporation (woks) Average

(ml)

amino group

I

2

3

4

0.90

0.95

0.96

0.94

72.2 incorporation:

No.

76.2

77.0

75.4

75.5 f 2.1’ pmollg

0 Aminopropyl glass (66.7 mg) was used for each determination. 0 The Hg(NO& titrant was standardized with KCI and was found be 0.00535 M in Hgl*. c Standard deviation.

to

QUANTITATIVE

2006

DETERMINATION



I

Titrant

OF AMINO

’ 2 volume,



’ 3

63

GROUPS



ml

FIG. 1. Duplicate potentiometric titrations of 958 mg of aminopropyl glass with 0.0514 A4 HCIO, in acetic acid. Two separate titrations are indicated by closed and open circles, respectively. The arrow indicates the equivalence point of 1.42 ml.

siderably smaller sample sizes were used compared with those used in analyses of Merrifield resins (8). This choice reflects accurately the peptide degradation experiment in which glass samples of small size may be utilized. In view of these two factors, the observed precision of about 3% is satisfactory and is in the range one would expect from titration results on Merrifield resins. Underivatized glass (HN03 treated and thoroughly washed) was subjected to picrate analysis which yielded a background value of 0.18 pmol/g. A titratable blank attributed to a glass reaction vessel has been noted in work with Merrifield resins (8). A sample of aminopropyl glass (not that used

in the studies described in Tables 1-4) containing by both picrate and aldehyde tests 121 -+ 3 pmol/g of amino groups was subjected to exhaustive treatment with glutaric anhydride (two successive treatments with 1 M glutaric anhydride in pyridine). Following these treatments the glass registered an amine content of 0.95 PmoYg. These results show that the species which give rise to the observed positive incorporation effects analyzed by the picrate or aldehyde methods (and by presumption, the other methods described here as well) are indeed the amino groups of the aminopropyl side chains. One has no primary standard by which one may gauge the “true” amine incorporation on glass supports. However, if a variety TABLE

4

COMPARISON OF METHODS FOR THE DETERMINATION OF AMINO GROUPS ON AMINOPROPYL GLASSY

300:

cl

2 Titrant

volume,

3 ml

FIG. 2. Potentiometric titration of 1.0 g of aminopropyl glass with 0.0564 M trifluoromethanesulfonic acid in acetic acid. The arrow indicates the equivalence point of 1.40 ml.

Method

Amino group incorporation (wnolk)

PIcrate test Aldehyde test Nonaqueous potentiometric titration Chloride ion titratton

16.1 k 2.7 78.7 * 2.7 76.2ta 76.2.6 78.Y 75.2 f 2.1

Time required 67 36 958 loo0 67

I5 min 16 hr I hr 20 min

u Aliquots of the same batch of glass were used in all determinations compared m thbs table. ’ HCIO, m acetic acid (0.0514 M). ’ CF,SO,H in acetic acid (0.0564 MI.

64

LEE AND LOUDON

of different techniques give self-consistent results, one can be more confident that systematic errors characteristic of a particular method of measurement are not being introduced. The first conclusion from Table 4 is that, within the precision of our measurements, all methods of measuring amine incorporation appear to be self-consistent, From the point of view of sample size and time required, the picrate method and Hg(NO& titration (once titrant is made up and standardized) are about equally convenient. The aldehyde test suffers from the time required for complete reaction; furthermore, we have found that the test is particularly sensitive to the thoroughness of the washing procedures. The nonaqueous titration method requires larger sample sizes; samples in the size range of those used for the other tests did not give reproducible titration curves using this method. However, in cases in which larger sample sizes are available, this method seems to work reasonably well. The inadvisability of using anhydrous HC104 for safety reasons should again be noted in the previous section. Trifluoromethanesulfonic acid (triflic acid) would appear to be a reasonable substitute for HClOJHOAc. The chloride ion titration, appears not to have been used extensively in analytical work on the Merrifield resin. This technique, although perhaps less convenient from the point of view of automation, might well prove useful in cases in which nonspecific absorption of picrate may be suspect and a rapid, independent amine test is required. ACKNOWLEDGMENT We gratefully acknowledge support of this work by the National Institute of General Medical Sciences.

REFERENCES 1. Merrifield, R. B. (1963) J. Amer. Chem. Sot. 85, 2149-2154. 2. Merrifield, R. B. (1964) J. Amer. Chem. Sot. 86, 304. 3 Esko, K., Karlsson, S., and Porath, J. (1%8) Acta

Chem.

Stand.

22, 3342-3344.

4. Brunfeldt, K., Roepstorff, P., and Thomsen, J. (1969) Acia Chem. Stand. 23, 2906-2907. 5. Dorman, L. C. (1969) Tetrahedron Lett. 23192321. 6. Gisin, B. F. (1972)Anal. Chim. Acta 58,248-249. 7. Tometsko, A. M., Schreiner, M., and Comstock, J. (1975) Anal. Biochem. 67, 182-191. 8. Hodges, R. S., and Merrifield, R. B. (1975) Anal. Biochem.

65, 241-272.

9. Schmitt, H. W., and Walker, J. E. (1977) Fed. Eur. Biochem. Sot. Lett. 81, 403-405. 10. Parham, M. E., and Loudon, G. M. (1978) Biothem. Biophys. Res. Comm. 80, 7-13. 11. Parham, M. E., and Loudon, G. M. (1978) Biothem.

Biophys.

Res. Comm.

80, 1-6.

12. Wachter, E., Hofner, H., and Machleidt, W. (1975) in Solid Phase Methods in Protein Sequence Analysis (Laursen, R. A., ed.), pp. 31-46, Pierce, Rockford, Ill. 13. Pryde, A. (1974) J. Chromatrogr. Sci. 12, 486478. 14. Grushka, E., and Kikta, E. J., Jr. (1977) Anal. Chem. 49, 1004A-1014A. 15. Willarmet, P. A., and Miller, J. G. (1976) J. Phys. Chem.

80, 2473-2477.

16. Fritz, J. S., and Schenk, G. H. (1974) Quantitative Analytical Chemistry, 3rd ed., pp. 195201 and 574-575, Allyn & Bacon, Boston. 17. Fritz, J. S. (1973) Acid-Base Titrations in Nonaqueous Solvents, p. 47, Allyn & Bacon, Boston. 18. Lane, E. S. (1961) Talanta 8, 849-852. 19. Muse, L. A. (1972) J. Chem. Educ. 49, A463A465. 20. Lalancette, R. A., and Steyermark, A. (1974) J. Ass.

Off.

Anal.

Chem.

57, 26-28.

21. Vogel, A. I. (1961) A Textbook of Quantitative Inorganic Analysis, 3rd ed., pp. 274-275, Wiley, New York.

Quantitative determination of amino groups on derivatized controlled pore glass: a comparison of methods.

ANALYTICAL BIOCHEMISTRY 94, 60-64 (1979) Quantitative Determination of Amino Groups on Derivatized Controlled Pore Glass: A Comparison of Methods C...
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