Reversed Phase Liquid Chromatography and Its Application

Reversed Phase Liquid Chromatography and Its Application to Biochemistry

Most analytical chemists are well aware of the rapid growth of high-performance liquid chromatography (HPLC) over the past 5-10 years. ANALYTICAL CHEMISTRY has recently published several articles in the "A" pages on HPLC topics (1-3). In addition, a number of recent books cover this subject (4-7). HPLC has proved to be a broadly applicable technique for separation and analysis in fields as diverse as environmental, pharmaceutical, and polymer chemistry. One of the latest fields to profit from the impact of HPLC is biochemistry. Although the contribution of HPLC to the other fields generally complements that achieved by GC, in biochemistry HPLC clearly is destined to play a much greater role. This is because so many biomolecules, due to their molecular complexity or large size, are thermally unstable or nonvolatile, preventing or complicating their analysis by GC. Among all of the factors contributing to the surge of interest in biochemical LC, none stands out as prominently as the development of reversed phase high-performance chromatography using n-alkyl chemically bonded phases (8,9). Thus, it is appropriate for this review to emphasize this mode [i.e., reversed phase liquid chromatography (RPLC)], although other modes will also be mentioned. In reversed phase, the stationary phase (typically hydrocarbonaceous) is less polar than the mobile phase (typically water/ methanol or water/acetonitrile mixtures). Substances thus elute in a general order of decreasing polarity, and mobile phase strength increases with decreasing polarity (e.g., acetonitrile is a stronger solvent than water). We shall first discuss current RPLC column performance and what can

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1048 A · ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978

reasonably be expected over the next several years. Next to be considered is the very important role of the mobile phase in the achievement of separation, an area where rapid advances are currently in progress. We will then shift to other HPLC developments that have or will have a significant impact,on biochemical LC, with special emphasis on trace analysis. Finally, the conclusion covers a few of the emerging applications that are beginning to have or will have a significant impact in biochemistry. Throughout this review, we try to provide a critical assessment. Reversed Phase Liquid Chromatography Already a significant majority of biochemical LC separations are performed by RPLC using bonded phases and water/organic mobile phases (814). Bonded phases are typically made by reaction of the appropriate chloro or alkoxysilane with a fully hydroxylated silica gel. Since this topic has recently been reviewed in ANALYTICAL CHEMISTRY

(3), we will only briefly

describe such phases. Equation 1 shows in simplified form a typical chlorosilane bonding reaction with a silanol group on the silica gel surface.

—^SiOH + ClSi(CH;()2R -^SiO—Si(CH:1)2R + HC1

(1)

For steric reasons (15), it is not possible for all silanol groups to react; indeed, depending on the chain length of R only about 45% will be bonded. 0003-2700/78/A350-1048$01.00/0 © 1978 American Chemical Society

Report

Barry L. Karger and Roger W. Giese Institute of Chemical Analysis Northeastern University Boston, Mass. 02115

Table I. Characteristics of Bonded Reversed Phase LC for Biochemical Separations

T h e silica surface then will consist of a mixture of silanols and hydrophobic chains, such as

R

R

I

I

CH 3 —Si—CH :i

C H — Si— CH:i

Ο

OH

Ο

I

I

I

— S i — O — S i — O— S i More silanols can be removed by t r e a t m e n t with trimethylchlorosilane, a small silylating agent. As a result of the bonding reaction, a hydrophilic surface is converted to an essentially hydrophobic one, consisting of a hydrocarbonaceous layer. Commercial materials are typically composed of R = ethyl, octyl, or octyldecyl (ODS); however, in principle, any chain length, e.g., C22 (16) or higher, can be employed. From a mass transfer point of view, a monomeric layer consisting of "bristles" of alkyl chains is general ly preferred over a polymeric layer t h a t can swell and undergo penetra tion by molecules, resulting in slow diffusion (12, 17, 18). Reaction with monochlorosilanes as in Equation 1 (rather t h a n the dichloro or trichloro analogs) is thus preferred, where poly merization cannot occur. In addition, incomplete reactions with di- and tri chloro analogs result in formation of silanol groups upon hydrolysis of unreacted silanol groups (11). T h e role of the n-alkyl chain length in separation is not fully clear at the present time. Under otherwise con s t a n t conditions, retention increases with chain length (10, 14) and column capacity is also greater with the longer

chain length. With regard to selectivi ty, there appears to be some differ ences as chain length is modified (19); however, in general, the differences are not as great as the retention change (8, 9). Indeed, control of selec tivity via modification of the mobile phase and use of a given bonded phase seems to be the approach adopted by most workers. Because of the protection of the underlying silica surface, a longer chain length tends to be more stable. Moreover, a longer chain length, e.g., Ci8, would be the selected phase for preparative scale work. T h e retentive ability of this phase can also be useful in the separation of weakly hydrophil ic substances by means of nonaqueous reversed phase chromatography (20). Countering these points is t h a t the shorter chain length phases, in our ex perience, lead to faster mass transfer and better efficiency. Indeed, Knox and Pryde found even better mass transfer on a short chain length bond ed phase than on the bare silica gel it self (12). It would thus appear t h a t the selection of the given phase involves a compromise between speed and ca pacity. For this paper, however, we shall ignore the chain length differ ences. Table I summarizes the positive fea tures of R P L C , particularly as they re late to biochemical LC, and helps ex plain why this mode is most frequent ly utilized even when the same analy ses can be achieved on unmodified sil ica gel itself. R P L C is also not without its problems, and Table I also lists current limitations, some of which may ultimately be overcome. We con sider first t h e positive features. T h e first favorable aspect of R P L C is t h a t a partially or fully aqueous mo bile phase is used. Most biomolecules

Positive Features Partly or fully aqueous mobile phase Compatibility with biological substances Operational simplicity Selective chemical equilibria Special detection modes Weak surface energies of bonded alkyl phase Rapid analyses Rapid mobile phase changes Trace enrichment High-performance columns Efficiency Selectivity Physicochemical tool for characterization Hydrophobicity Complexation equilibria Definition of purity Limitations Limited pH range of bonded phases (~2-7.5) Unreacted and accessible silanol groups, retention of basic substances Incomplete understanding of retention Shortcomings of current commercial columns Stability Repeatability of retention and selectivity Column efficiency

are compatible with such an environ ment; thus, solubility can be readily achieved. Moreover, because the water content of the mobile phase can range from 100% to quite low percentages (or none at all), a broad spectrum of biomolecules can be chromatographed, e.g., lipophilic or ionic, small or large. T h e ability to separate a wide range of substances of varying hydrophobicities (perhaps with a solvent gradient) has obvious application when complex mixtures are to be ana lyzed. An example of this is shown in Figure 1 in the separation of urinary acids using TJV detection (21). In fact, the frequent success of simple mobile phase compositions in achieving sepa-

ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978 · 1049 A

Figure 1. Fluorescence and UV absorbance chromatograms of urine containing added aromatic acids Sample: 10 mm 3 of urine extract with about 100 ng of each acid. Numbers refer to individual acids cited in article. Conditions: Column, S μπ\ LiChrosorb RP-18, 25 cm long: flow rate, 2 mL/min; temperature, 70 °C; gradient elution from 0.1 mol d m - 3 phosphate buffer, pH 2.1 with acetonitrile; initial Inlet pres sure, 160 bar. Fluorescence detector, excitation 238 nm, emission 340 nm, time constant 0.S s Reprinted with permission from rel. 21. Copyright 1978 Pergamon Press


ration, along with other factors listed in Table I, has led to an overall opera tional simplicity for RPLC. The mobile aqueous/immobile lipo philic system of RP also allows selec tive equilibria such as ionization (22, 23), ion pair (24, 25), and ligand ex change (26) to be employed, creating special separation opportunities for ionizable substances. Further, the RPLC mobile phase system permits special opportunities using electro chemical detection (2), and postcolumn reaction detection in aqueous media (27). Finally, there are subtle advantages relating to column repro ducibility. For example, water in the injection solvent will not in general af fect retention, because of the presence of water already in the mobile phase. In contrast, traces of water in the in jection solvent can at times cause ret ention variation in the case of silica gel. A similar advantage is that highly polar (e.g., ionic) interferences fre quently elute unretained in RPLC, whereas they accumulate on the col umn, gradually degrading the column performance, on bare silica packings. The next feature resulting in the popularity of RPLC is the weak sur face energies of the bonded alkyl phase (15), permitting not only rapid analyses but also rapid reequilibration when mobile phases are altered. Typi cally, only 5-10 column volumes are necessary for equilibrium to be reached vs. often 50-100 or more vol umes for silica. Solvent scouting for optimum resolution is therefore rapid, and return to starting conditions in gradient elution is generally fast. For the same reason, aqueous solutions can be injected directly into RPLC columns under conditions of an aque ous/organic mobile phase. This tech nique of trace enrichment allows preconcentration of the analyte at the be ginning of the column (i.e., water is a very weak solvent) and thus allows particularly large volumes of sample to be delivered to the column (28). Not only are aqueous solutions inject ed, but urine or serum has also been injected directly onto η-alkyl bonded phase columns (29) or onto a precolumn (30). Another general advantage of RP for biochemical LC is the high column performance or resolving power. Not only is the RP system quite selective both toward hydrophobic and polar group solute differences (31), but high column efficiencies comparable to normal adsorption chromatography are possible (12). Finally, there is the expectation that RPLC using bonded phases will become an important tool in charac terization of biochemical substances, e.g., the measurement of fundamental properties of molecules such as hydro-

phobicity (32) and complexation constants in aqueous media (9). Hydrophobic characterization using TLC or paper chromatography has, of course, been extensively studied (33). Moreover, as RPLC is a separation tool, analysis by this mode could be used as a definition of purity, in much the same way as other chromatographic processes are employed. Let us next turn to a consideration of current limitations in RPLC. In general, there is a limited pH range (~2-7.5) over which stable columns can be maintained. At low pH, attack of the Si—C bond (see Equation 1) is possible, whereas at high pH, the silica matrix may be attacked, particularly in salt solutions (a frequent condition in biochemical LC). Some workers have performed separations outside this pH range, but the long-term stability (i.e., months of use) of the bonded phase is questionable. This limited pH range need not be a significant handicap, however, since in general most separations can be achieved in this range, at times using secondary chemical equilibria (e.g., ion pair chromatography). A second problem can arise from the remaining silanol groups (Si—OH) on the silica surface influencing the retention of polar, and particularly basic substances, e.g., amines. If these silanol groups are accessible for interaction, then retention will be affected, and peak tailing may result as a consequence of a mixed retention mechanism. A competing base is often added to reduce interaction and improve peak symmetry; alternatively, ion pair chromatography can be employed. A third limitation deals with our lack of understanding of the details of retention in RPLC. Initially, workers argued whether bonded phase retention could be viewed as partition (i.e., solution) or adsorption. It is now generally recognized that the retention process is more complicated and cannot be placed in one of the classical forms of chromatography. Moreover, the role of the organic modifier (e.g., methanol, acetonitrile) that is extracted into the stationary phase (19) is unclear. In addition, the use of ionic equilibria for control of separation can often be complex. From a practical point of view, it is important that we develop a better understanding of the retention process so that we can better control it. A fourth limitation relates to the quality of commercial RPLC columns currently available. In spite of the general acceptance of such columns, there is the growing awareness that their overall performance can be improved, particularly with respect to stability, reproducibility in retention and selectivity, and efficiency. With

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most manufacturers working actively in the area of column improvement (indeed, some companies have recent ly introduced new product lines) and with the knowledge available to over come many of the problems, we can predict that a new generation of com mercial RPLC columns is on the hori zon. In the next section we discuss the problems and suggest the characteris tics of the columns that are to be an ticipated. RPLC Columns. The central role of the column in LC is well appreciat ed. A great deal of effort has been ex pended in attempting to understand band broadening phenomena in LC (12, 34-38). We now have a fairly good appreciation of the compromises that must be struck with respect to theo retical plates, time and pressure, and the central role of particle diameter on column performance (39-41). Smaller particle sizes lead to faster analyses (34). Most columns now con sist of fully porous particles in the 510-μπι diameter range and come in lengths of 15-25 cm. There is a defi nite trend toward the use of 5 μια, be cause such particles lead to faster analyses and are no more difficult to pack than 10 Mm (contrary to what was originally believed). It is unlikely that columns with par ticles of dp (particle diameter) much less than 5 Mm will become generally available because of theoretical and practical problems. The low perme ability of such columns with very small dp means that at the maximum pump pressure (i.e., constant pump pressure) the mobile phase velocity will decrease with decreasing dp. Eventually, column efficiency will di minish with lower dp (at the maxi mum pump pressure), since the veloc ity will be less than the optimum value (42). This limit in dp appears to be ~2-3 Mm at 6000-8000 psi pump pres sure. Moreover, Horvath and Lin have recently claimed that kinetic processes of stationary/mobile phase transfer may also become limiting at very small particle size (43). From the practical side, column length is typically decreased at the same time dp is lowered, and heavy demands are thus placed on the mini mization of extra column effects, e.g., injection, connection tube, cell vol ume, and response time of the detec tor. These extra column demands may be surmountable but at a loss at present in convenience (44). Another problem can arise from thermal ef fects caused by frictional forces associ ated with flow in the small particle columns (45). Finally, it is important that dp is reproducible from batch to batch and that the particle size is nar rowly distributed. Efficiency and col umn permeability are sensitive dp

1052 A · ANALYTICAL CHEMISTRY, VOL. 50. NO. 12, OCTOBER 1978

functions that are magnified at small dp's. For all of the above reasons, col umns with dp ~ 5 Mm appear to repre sent a good compromise for the fore seeable future with respect to perfor mance and ease of manufacture (which is related to cost). A question often raised is whether to use irregular or spherical-shaped particles. Both types are commercially available in both 5- and ΙΟ-μιτι sizes and have been extensively employed. While both give roughly similar plate counts, spherical particles are general ly preferred because of their higher permeability (46) and their potential ly better column packing stability (at least when slurry packing techniques are followed by bed compaction). We have noted previously that a current problem is the instability of the commercial RPLC columns. As the columns are expensive ($300), this is of particular concern. But if a prop er packing procedure is followed and proper care is exercised, columns should be stable for 3-6 months or more with continual use. A poorly packed column can lead to a resettling of the packing with use, creating a void at the top of the column. Since this is empty space in which sample is injected, broad peaks with very poor symmetry can result. Corrective pro cedures such as filling in the void with either glass beads or packing have been recommended; however, with proper packing techniques such voids should seldom occur. As has been de scribed in the literature by Kirkland (13), slurry packing followed by col umn compaction via pressure pulsing substantially reduces the instability problem. The experience of our labo ratory agrees with that of Kirkland. Instability can also arise from im proper operation and care of the col umn. As already mentioned, the pH should generally be within the range of 2-7.5, to prevent decomposition of the packing itself. In addition, col umns should be stored in methanol or methanol/water mixtures and not in aqueous buffers. The quality of the bonded phase is also important. To reduce attack of the silica, the appro priate monochlorosilane (see Equation 1) should be reacted to the greatest extent possible. Maximum coverage typically represents n-alkyl bonded phase surface concentrations of 3.3 Mmol/m2 or greater (11, 13). As noted, a silanization step with trimethylchlorosilane may also prove useful. Final ly, since small particle columns are ex cellent filters, it is important to re move particulates from mobile phases and, where appropriate, from sample solutions prior to injection. The reproducibility of retention and selectivity is also expected to improve (Continued on page 1057 A)

significantly in the next generation of RPLC commercial columns. It is not unreasonable to expect retention to be repeatable within 10% and relative retention of less than 5% from column to column with polar (but nonionic) substances (47). Reproducibility is first a function of the repeatability of the silica-based matrix with respect to surface area and pore structure. In our experience this is an area that is not sufficiently addressed by some manufacturers. The next factor is re peatability of the bonding reaction, as shown in Equation 1. This repeat ability is dependent on the extent of carbon content and perhaps more sig nificantly for some solutes, e.g., amines, a function of unreacted and accessible silanol groups. Accessible silanol groups can also affect selectivi ty of polar nonionic substances (47a). Repeatability from column to col umn is poorer when mixed retention mechanisms (in this case, hydrophobic and adsorption on silanols) occur vs. a single retention mechanism. Reten tion and relative retention are then improved by eliminating as much as possible the accessible silanol groups. This is best accomplished again by maximum coverage of the bonded phase. What can reasonably be expected from the new generation of RPLC col umns with respect to efficiency? These columns should be able to achieve reproducibly reduced plate heights h (h = H/dp; H = plate height, dp = particle diameter) be tween 3 and 4 for standard nonionic substances at reduced velocities of ν =i 10 (v = udp/Du; u = mobile phase velocity, DM = mobile phase diffusion coefficient of solute) and to achieve h at 10 at reduced velocities of 100. For 5-μπι particle sizes h at 3-4 trans lates to ~9000 plates for L = 15 cm and ~15 000 plates for L = 25 cm. Note that this is not a very stringent requirement, considering that re search laboratories are able to achieve h values of 2-2.25 or less (12). Peak symmetry has recently been advocated as an important criterion of column performance (48). Asym metrical peaks can result from extra column effects, particularly injection problems; however, these problems are solvable in principle. Asymmetry may also arise from a poorly packed column, and this is invariably the problem when standard substances (nonionics) are used with well-defined equipment (i.e., minimized extracolumn effects) (48). With a well-packed research column an asymmetry factor (As) (As = b/a where b and a are the peak half widths at 10% of the peak height) between 0.90-1.1 should be achievable with standard substances (49). A relaxation of specifications to

a maximum of 1.3 would therefore seem reasonable for large-scale col umn production. Because of the structural complexi ty or large size of many biomolecules, perfectly symmetrical peaks may not arise as a result of thermodynamic/ kinetic considerations of the distribu tion equilibrium. In general, some im provement in symmetry is obtained by an increase in temperature, a con trol of interfering chemical equilibria (24), or a change in mobile phase ve locity (50). In summary, RPLC columns of high stability, consistent retention (abso lute and relative), and high efficiency are feasible and should be routinely available in the near future. With these developments, HPLC will be come less of an art and more of a science. Mobile Phase Control of Selec tivity. The separating power of RPLC arises not only from the quality of the columns but also from the control of the mobile phase. A great deal of ac tivity is currently underway in an at tempt to understand the retention process in RPLC and predict selectivi ty, and expand the control of selectivi ty via the incorporation of chemical equilibrium steps (e.g., ionization). We shall now describe several aspects of these developments that appear to be most relevant to biochemical LC. As we have noted, the detailed mechanism of retention with chemi cally bonded η-alky 1 stationary phases is in question (8,19). Nevertheless, the central role of hydrophobic (or solvophobic) phenomena (52 ) is not in doubt (31, 52, 53). Here, as a conse quence of the very high cohesive ener gy density of the solvent (arising from the three-dimensional hydrogen bond ed network), solutes are "squeezed out" of the mobile phase and are bound with the hydrocarbon ligands of the stationary phase. (Note the driving force for retention in this case is not the favorable interaction of so lute with stationary phase, but the ef fect of solvent in forcing the solute to the hydrocarbonaceous layer). As this phenomenon is opposed by polar group interaction of the solute with the mobile phase, hydrophobic reten tion (or relative retention) deals main ly with nonpolar substances (e.g., aro matic hydrocarbons) or the nonpolar portions of molecules. Hydrophobic selectivity arises as a consequence of nonpolar size differ ences (i.e., nonpolar surface areas) of solutes (54, 55). Thus, selectivity based in part on molecular size is readily achievable. Good resolution of homologs, for example, is observed, with improved separation as the water content of the mobile phase is in creased, for a given stationary phase.

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Figure 2. Separation of fat-soluble vitamins Column: Waters Associates μ Bondapak, 30 cm X 4 mm in A and C, 60 cm X 4 mm in B. Chromatographic conditions on figure. A-Ac = vitamin A acetate, D 2 = vitamin D2. I = interference, Ε-Ac = vitamin Ε acetate

That RPLC is a good discriminator of nonpolar portions of molecules was already known in the earlier studies on reversed phase paper and thinlayer chromatography (56). Recently, a better appreciation of the power of RPLC to resolve func tional group differences has emerged. The central role of the organic modifi er has become clear, and polar group selectivity has been shown to be quite dependent on the organic solvent cho sen (47a, 57). This appreciation has led to the use of ternary mobile phases consisting of H2O and two organic modifiers, e.g., MeOH and THF, for the precise con trol of selectivity of solutes particular ly with different functionalities. Fig ure 2 illustrates this approach with an application from our laboratory (58), involving the separation of fat soluble vitamins. In Figure 2A an interference (known to possess a carbonyl group) overlaps completely with vitamin D2 (which contains an alcohol group) with 95/5 (v/v) MeOH/H 2 0. With a longer column and separation time, this interference is partially resolved, as shown in Figure 2B (a =* 1.03), but addition of THF to change the mobile phase into a ternary system leads to a baseline separation (« =^ 1.25) on the original 30-cm column in only 4 min, as shown in Figure 2C. Efforts are now underway to understand and predict behavior, perhaps by a multi parameter approach as employed for classifying phases in GLC (59, 60). Since many biomolecules are multi functional, ternary phases would ap pear to have a good future in this field. The use of chemical equilibria im posed on top of the physical distribu tion process has received increasing attention in RPLC for control of ret ention and separation. The concentra tion and/or form of the solute is pur posely altered in the process. As a con sequence of aqueous/organic mobile phases, chemical equilibrium involv ing ionic species is a particularly fruit ful area of study. The most obvious example is pH control of the mobile phase, an approach already well

known in classical RPLC, ion ex change, and other ion equilibria sys tems (61 ) . Here, the solubility of the substance in the mobile phase is al tered by the extent of its ionization. As a general rule, it is preferable to chromatograph substances in their nonionized form, since there is then less chance for secondary equilibria influencing retention or band shape. The pH approach is generally limit ed by the instability of the bonded phases outside the pH range of 2-7.5. Thus, ionization control of strong acids and bases by this approach is not possible. Moreover, as we have noted, protonated species (i.e., strong bases) can adsorb on accessible silanol groups leading to peak tailing. A special mode that moderates or eliminates the above problems and also controls selectivity is reversed phase ion pair chromatography (25, 62, 63). Here, retention of ionic species is determined by adding counterions in order to neutralize the charge of the solute, as in the equilib rium below Aaq + B aq

(A, B) 0

(2)

where aq, org = aqueous and organic phases, respectively, and where either of the ions on the left-hand side is the solute and the other is the counterion. Typical counterions include alkyl sul fates or sulfonates, and tetraalkylammonium salts. The chain length of the counterion controls the hydrophobicity of the final ion pair product and thus the extent of retardation (or ac celeration) of the solute. In principle, both pH and counterion effects can be important, for both the solute and the exposed silanol groups. Typically, relatively short alkyl chain lengths, e.g., hexane sulfonate, are used. However, chain lengths as high as 12-16, i.e., detergents, have been at times selected (64, 65). In this latter case a significant amount of sur factant (in excess of 10 mg/g of sup port) is adsorbed onto the bonded phase. This mode has been called soap (Continued on page 1062A)


Figure 3. Soap chromatography of cat echolamines and derivatives Column: 12.5 cm X 5 mm i.d., 5 μπι spherical silica with octadecyl/trimethyl bonding; eluent, water-acetonitrile-sodium lauryl sulfate-sulfuric acid 70:30:0.02:0.04 (v/v/w/v); detection, 280 nm, 0.01 aufs. Solutes: NA = noradrenaline, LD = L-DOPA, A = adrenaline, NMA = normetadrenaline: DA = dopamine, MA = metadrenaline, MD = «-methyldopa Reprinted with permission from ref. 67. Copyright 1976 Journal of Chromatography

chromatography or dynamic ion-ex change chromatography (66). An ex ample of a rapid separation by this mode is shown in Figure 3, involving a series of catecholamines (67). We can anticipate the use of reversed phase ion pair chromatography as a substi tute for ion-exchange chromatography using organic resins or bonded ion ex changers (stability problem) in the fu ture. Since ion-exchange is a signifi cant separation mode in biochemical LC, the ion pair mode would appear to have great potential, especially for an alytical purposes. Typically, reversed phase ion pair chromatography, as currently prac ticed, takes advantage of differences in the hydrophobicity or hydrophilicity of the substances themselves. With the use of the simple counterions, the ion pair step involves mainly an electrostatic interaction. Obviously, the use of more complex counterions can contribute further selectivity based on differences in which differ ent ionic solutes interact with the counterion. An example of this approach is shown in Figure 4 in the isocratic sep aration of dansylamino acids using as counterion Ci2-dien + Zn(II)

In this case, the metal chelate repre sents a conformationally semirigid structure with a local polarized charge center. As such, not only is there the typical electrostatic attraction of an ions for the positively charged chelate, but also steric effects and hydrogen bonding may occur. Such chelates can lead to high selectivity. For example, the separation of fumaric vs. maleic acid (cis vs. trans dicarboxylic acid) occurs with a relative retention of 2.5 (vs. 0.90 with a reversed phase ion pair system), and 2,4-dichlorobenzoic acid vs. 3,5-dichlorobenzoic acid gives a = 7.8 (vs. 2.8 with a reversed phase ion pair system). It is obvious that the structure of the metal chelate can be tailored for favorable interaction with certain solutes, thereby providing spe cific control of selectivity. Details of this approach can be found in two re cent articles (68, 69). The metal chelate approach illus trates one way in which metals can be used to develop special selectivities in RPLC. A second approach is to add the metal directly to the mobile phase. A solute that complexes rapidly with the metal ion will then be made gener ally more hydrophilic and thus elute more rapidly from the column. As an

Figure 4. Separation of dansylamino acids by Ci2-dien-Zn(ll) chro matography

Conditions: 1 0 - 3 M ZnS0 4 ; 1 0 - 3 M C 12 -dien; 1 % ammo nium acetate; acetonltrllewater (35/65); 25 cm X 4.6 mm i.d., Merck LiChrosorb C e . Solutes: 1. glutamic acid; 2, 7-aminobutyrlc acid; 3, threo nine; 4, serine; 5, «-aminobutyric acid; 6. norvaline: 7. leucine; 8, tryptophan Reprinted with permission from ref. 6β. Copyright 1978 Journal of Chromatography

example, Sternson et al. added Ni(II) to the mobile phase to selectively ac celerate ortfto-aminophenol relative to other phenolic substances (70). Perhaps the most popular approach in this area is the addition of Ag(I) to the mobile phase for selective charge transfer complexation with unsatu rated centers on molecules. This ap plication of argentation chromatogra phy is useful for taking advantage of cis vs. trans, unsaturated vs. saturat ed, and heterocyclic differences among solutes, as has been the case classical ly. An excellent summary of work in argentation RPLC can be found in a recent paper (70). Trace Analysis. A major goal in biochemical LC is detection at low concentration levels, as low as pg/mL in solutions or physiological fluids. A great deal of effort has therefore been expended in examining the various factors that control the optimization of LC for trace analysis. There are ba sically four areas of interest: LC col umn and conditions, injection solvent, detector, and pre- and postcolumn derivatization. By a judicious selection of technique, chemistry, and detec tion, ultratrace analysis at the picogram level is now possible, with the very real hope that femtogram (10~15 g) detection will be achieved in the not-too-distant future. We shall now examine briefly the four areas. It is not often recognized that a chromatographic column is a dilution device, with the greater dilution the larger the retention. Thus, the sample starts off as a (relatively) concentrated mixture and may be eluted from the column diluted ~10-100-fold or more by the mobile phase. This extent of dilution need not occur. Indeed, by appropriate control of chromato graphic conditions only a ~3-5-fold

1062 A · ANALYTICAL CHEMISTRY. VOL. 50, NO. 12, OCTOBER

1978

dilution may be all that need take place depending on the separation problem. Optimization of the chroma tographic column for trace analysis was treated several years ago by Huber et al. (71), Kirkland (72), and Karger et al. (73). As a general conclu sion, high-performance fast columns are desirable for trace analysis, along with k' (capacity factor), where possi ble, less than 2. A particularly critical parameter is the volume of sample injected into the column. Consider using the same sol vent for the sample and mobile phase. There are two significant points to be noted. First, for a given column diam eter, much larger sample volumes can be injected into LC than, for example, GC columns. It is not at all unusual to inject 100 μL· or more into an LC col umn. This, of course, means that for a given solute concentration, more sam ple can be delivered to an LC column for trace analysis. This advantage translates mainly into a convenience since the sample may have to be con centrated for GC injection. However, there may be problems with solubility of major components in the matrix, in which case concentration procedures may be limited. Secondly, it is often assumed that narrower bore columns are better for trace analysis than wider bore, since sample dilution is less. It must, how ever, be emphasized that this is only true if the sample is limited to very small amounts, e.g., a few microliters. If an optimum or maximum sample volume is defined as that producing a certain percentage increase (e.g., 10%) over the band width (i.e., variance) of the peak caused by the column, it can be shown that peak dilution is inde pendent of tube diameter (71, 73). The point is that a larger volume of

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sample (proportional to the square of the tube diameter) can be injected into the wider bore column. The use of the recently developed microbore col umns (44) (e.g., tube diameter of 0.5 mm) for trace analysis should be viewed in this light. A natural extension of the large sample volume technique is to use an injection solvent that is significantly weaker than that of the mobile phase. In this case, a preconcentration of the solute will occur at the top of the col umn, as the mobile phase at the begin ning of the column is converted to the weaker injection solvent. Historically, this preconcentration approach was well known and applied in tempera ture programming GC and solvent programming LC. With reversed phase columns, the injection of aque ous solutions has become quite popu lar for' trace analysis, and the general technique is called trace enrichment. The approach has been especially popular in pollution analysis (74), where very large volumes (e.g., hun dreds of milliliters) have been injected into reversed phase columns, followed by gradient elution. Trace enrichment is finding increas ing use in biochemical LC. One report by Frei and coworkers showed that quantitative trace analysis of pep tides, e.g., oxytocin, is possible by this approach, where almost 2 mL of an aqueous buffer solution (28) were in jected. In a very recent paper by Lankelma and Poppe (75), an analysis of methotrexate in serum was set up for routine use involving a dual-col umn trace enrichment procedure. An RPLC column was used first for pre concentration, followed by an ion-ex change separation column, permitting detection at the ng/mL level. While trace enrichment clearly has a great potential in biochemical LC, neverthe less, one must recognize that interfer ences will also tend to concentrate along with the solutes, and sample vol umes of physiological fluids may ulti mately be limited. The sensitivity of the detector plays a very significant role in the ultimate limits of an analysis. Thus far, the de tectors that have had the most signifi cant impact in LC trace analysis have been UV absorbance, fluorescence, and electrochemical. A notable appli cation of absorbance detection is the recent, direct sequencing of nanomole amounts of protein involving the LC detection of PTH-amino acids at 254 nm (76, 77). The sequence of the Nterminal 47 residues of sperm whale myoglobin was determined starting in one case with only 0.2 nmol of materi al (76). Although much greater sensi tivity for the analysis of amino acids can currently be achieved by GC (78), alternative derivatives are involved in NO. 12, OCTOBER

1978

this case which are less directly appli cable to sequencing procedures. Electrochemical detection for the right set of samples is a useful ap proach to pg analysis (2, 79). Both the amperometric and coulometric modes of detection are employed (79, 80). In general, lower levels of detection are possible in the oxidation mode than the reduction mode; therefore, most applications at the trace level have in volved oxidation, e.g., catecholamines. This detection mode is finding, for ex ample, use in the analysis of blood/ brain barrier metabolic processes (81). A particularly promising approach is the use of electrochemical detection following postcolumn redox couples (82). For example, sugars can be de termined by using the Fe(CN)jf4/ Fe(CN)ë s couple with detection via the oxidation of Fe(CN)g4 (83). Unfortunately, electrode fouling can be a problem. Probably the detector that currently holds the greatest promise for ultratrace analysis of compounds of biochemical interest at present is fluorescence. A moderate fraction of the analytes of interest either is inherently fluorescent (e.g., porphyrins, many of the vitamins, several drugs), or can be made so by derivatization before or after the column (e.g., amino acids, catecholamines, polyamines, peptides, proteins, several drugs). Commercial fluorescence LC detectors can quantitate at levels as low as 10-20 pg in favorable cases (e.g., excitation of dansyl derivatives at 340 nm) (84). A particularly promising approach on the horizon for ultratrace analysis (i.e., femtogram level) is the use of high-intensity laser sources. (In fluorescence the signal is proportional to the source intensity.) Zare detected 750 fg of aflatoxins by this approach (with greater than 100-fold dilution in the column) (85). Ultimate detection limits in fluorescence, or in any detection system, will depend on our ability to purify solvents as mobile phases. New demands on solvent purity will undoubtedly add cost to the mobile phases, and microbore columns, which have flow rates of only ^L/min, may thus become of significance. An often cited advantage for HPLC, relative to GC, is that derivatization is not essential for chromatographic analysis of many specific solutes. While this is true, workers are finding increasing value in pre- and postcolumn derivatization with respect to either selective and/or trace detection (86). Classically, this approach has been followed in many biochemical LC analyses, the most prominent being postcolumn ninhydrin detection of amino acids (87, 88). Postcolumn detection has attracted increasing interest in the last several years for the

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1978

trace analysis of such biomolecules as the isoenzymes of creatine kinase and lactate dehydrogenase (89), creatinine (90), polyamines (91 ), digoxin (92), and indoles (93). We include in postcolumn reaction detection those cases where a reactant and analyte produce a derivative of different detection properties than either of the starting materials, e.g., fluorescamine reacting with solutes containing primary amino groups (27), as well as reaction couples, e.g., redox reactions, where the product of the analyte or the reagent is detected, e.g., the previous cited example of Fe(CN)ëV Fe(CN)ë 3 redox couple for detection of sugars. Most effort to-date has been directed in the design of reactors in order to minimize band spreading in postcolumn detection. Three general approaches are in current use, dependent on the reaction time. [Importantly, 100% conversion of reactants to products is not necessary; the major need is reaction reproducibility (94).] In the first, which is useful for reactions of the order of 1 min or less, the reactant and column effluents are mixed in an appropriately designed tee and allowed to react in a coil. For reactions of ~ l - 5 min duration, packed beds are used (95), whereas for reactions from 5 to 20 min, air segmented flow is the best approach (96). Above ~20 min, effluent storage can be used (97). Here, air segmented streams are stored in reaction coils for extended periods of time and then brought to the detector. Many instrument companies are working on reaction detectors; we expect commercial reactors will be widely available. Precolumn derivatization will no doubt as well continue to be widely applied to LC trace analysis, e.g., dansyl amino acids (98). A major advantage over postcolumn reaction detection is that the product need not have different detection properties from the reactant or analyte; different separation properties are adequate. Moreover, somewhat greater freedom is available in the selection of precolumn derivatives, since long reaction times can be allowed. In postcolumn derivatization such times may degrade separation via an increase in band width. Finally, it is easier, for a small number of analyses, to set up the derivatization manually prior to the column rather than to develop an on-line system suitable for postcolumn reaction detection. Nevertheless, some contrasting advantages for postcolumn reaction detection derivatization can also be important. First, there is much less need to convert quantitatively each analyte into a single product than in precolumn work. Secondly, precolumn derivatives make the so-

lutes more similar, potentially creating a separation problem for some applications. Thirdly, the postcolumn reaction occurs in a relatively constant environment, whereas a varying environment may occur in the precolumn case. Finally, postcolumn derivatization can be readily automated, an important consideration for the high-volume assays found in the clinical laboratory. (Precolumn derivatization can also be automated, but often less conveniently.) Applications of Biochemical LC

As we have noted much of the current activity in the application of HPLC to biochemical problems involves the use of reversed phase columns. Since many of these applications are summarized in other sources (99-101), we shall only briefly comment on the current state of affairs. We shall then turn to selected biochemical LC areas, i.e., complex lipids, polypeptides, and proteins that are at an earlier stage of development. Each area promises to have a significant impact in biochemistry. Much of the interesting work on the complex lipids and polypeptides is now being conducted with RPLC, continuing the current trend in column use. Only in the case of protein separation is the development at present leading to different types of stationary phases. Prominent among the early applications of biochemical LC were analyses by RPLC of biogenic amines (e.g., catecholamines, indoles, polyamines), amino acids, nucleotides/nucleosides/ bases, porphyrins, and drugs. The analysis of the biogenic amines and amino acids was motivated in part by the good detection opportunities provided by either electrochemical detection (for the catecholamines) or the pre/postcolumn reaction detection (e.g., ninhydrin, o-phthalaldehyde, fluorescamine). Nucleotides/nucleosides/bases (102) and the porphyrins (103) were difficult separation problems for other methodologies because of their high polarity and numerous closely related forms. Thus, LC was quickly turned to as soon as it became available. The main reason for the LC analysis of drugs in physiological samples is therapeutic monitoring, although pharmacokinetic and metabolic studies are at times performed by LC. The purpose of therapeutic drug monitoring is to establish efficacy and avoid toxicity since, for many drugs, therapeutic and side effects correlate better with serum levels than with dosage. Among the most popular LC analyses thus far are those for the cardiac drugs, e.g., procainamide, the antiepileptics, e.g., phenytoin and phénobarbital, and theophylline, an antiasth-

matic. Although LC faces considerable competition from alternate methodologies, especially immunoassays and GC, LC already has made major strides and will continue to grow dramatically in this area. A recent review on clinical liquid chromatography has discussed these and other drug applications in detail (100). We next turn to emerging areas of biochemical LC. Complex Lipids. The use of LC for the separation and analysis of simple lipids such as fatty acids, steroids, prostaglandins, and fat-soluble vitamins is well established. However, more complex lipids such as glycosphingolipids have presented a continuing problem. Besides the difficulty of separating complex mixtures, e.g., multiple closely related forms differing only in chain length or double bond position, there is the problem of detection particularly with respect to sensitivity and quantitation. Here derivatization has played a significant role in overcoming some of the detection problems (104). Much of the early LC work was performed on silica gels using a moving wire detector, as summarized in the review of Aitzetmuller (105). More recently, RPLC has been actively pursued. Both methods have been found to complement one another quite well. In some cases, silica is a useful class separator, whereas RPLC resolves the


class. In other cases, the opposite is true. For example, McCluer recently observed that a single sphingomyelin peak on silica converted into 13 peaks on RPLC (106). On the other hand, Fitzpatrick found that the simple lipid, prostaglandin PGE2 (as its pbromophenacyl ester) could be maintained as a single peak on RPLC, but that adsorption chromatography on silica gave further resolution into its four geometric or epimeric isomers (107). In this case, the class separation on RPLC was considered to be more useful (i.e., single peaks for PGF 2 „, PGE2, PGD2) since the objective was to monitor the enzymatic biosynthesis of these prostaglandins by class rather than in a more detailed manner. Clearly, LC will be used increasingly to expand our knowledge of lipid biochemistry and to characterize these substances, especially by use of RPLC for the complex lipids. Peptides. Peptides are heterogeneous both in regard to their functions and structures. The functional heterogeneity arises from their importance as synthetic intermediates, hormones, pharmaceuticals, antibiotics, and protein degradation products. The structural heterogeneity derives from the presence of cyclic forms (especially for peptide antibiotics), the occurrence of diastereomers (involving enantiomeric

Figure S. Separation of angiotensin peptides Column: LiChrosorb RP-8 (5 μτη octadecyl-silica) 25 cm; gradient elution from 0.1 M phosphate buffer, pH 2.1, with acetonitrile as the gradient former; gradient shape, refer to article; Τ = 70 °C, flow rate = 2 mL/min. Numbers refer to various angiotensins Reprinted with permission from ref. 108. Copyright 1976 Academ ic Press

substitution), variation in the number of residues, and the extent and type of chemical modification (both on the side chains and at the carboxy and amino termini). In all of these cases, LC and espe cially RPLC potentially can be impor tant for the purposes of purification, assessment of purity, and character ization. High efficiencies and good re coveries are achieved using chemically bonded reversed phases, as illustrated in Figure 5, where a series of angioten sins and angiotensin analogs (peptides ranging in size from 5 to 10 amino acids) are separated by gradient elution {108). Several groups have been active in this area, including Molnar and Horvath (108), Krummen and Frei (109), Rivier (110), Hancock et al. (Ill), and Sasagawa and Okuyama (112). In gen eral, a common feature has been the use of a phosphate buffer on reversed phase columns, particularly at low pH ~ 2. Besides its high UV transparency at low wavelengths, it is claimed that phosphate may exert a favorable complexation ability with a peptide to re duce band asymmetry and raise plate count (111). Peptide RPLC separations have al ready been put to good use. For exam ple, oxytocin, a nonapeptide, has been separated by RPLC from (2-D-tyr)oxytocin (113). These two diastereoisomers differ in configuration (D vs. L) only in the amino acid at the 2 po sition. In another case, RPLC was used to provide a rapid (10 min) and sensitive separation of two diastereomers of fluorine-substituted somatos tatin (a tetradecapeptide). These ste reoisomers differed in configuration only at the 8 position (114). As a final example, RPLC resolved gramicidin, a pentadecapeptide antibiotic, into five peaks which preliminary data suggest are five conformers (108). If true, it may be possible to elucidate popula tions of conformers as a function of pH, temperature, and concentration of organic solvent. Of course, preparative scale purification of peptides will also be a significant development. These examples illustrate the bright future for LC in the study of peptides. Proteins. Column LC has been used for many years as a purification and analytical tool for protein mix tures, utilizing gel permeation, ionexchange, or hydrophobic chromatog raphy on organic gels, e.g., cellulose (61 ) . These packings are pressure un stable, and as a consequence only low flow rates and slow separations are possible. The extension of HPLC to high-speed protein separations using high pressures has been a goal for a decade or more. The search for pres sure stable packings that at the same time permit quantitative recoveries of 1070 A ·

Figure 6. Separation of CPK isoen zymes using postcolumn enzyme de tector Column, 25 cm Χ 4 mm i.d.; DEAE-glycophase/ CPG (250 A pore diameter. 5-10 μπι); solvents (gradient) A = 0.05 M Tris. 0.05 M NaCI, 1 0 - 3 M mercaptoethanol, pM 7.5, Β = 0.05 M Tris, 0.3 M NaCI. 1 c r 3 M mercaptoethanol, pH 7.5; flow rate 3 mL/min; a = CPK3, b = CPK2, c = CPK, Reprinted with permission from ref. 89. Copyright 1976 Journal of Chromatography

proteins without denaturation has only met with minimum success. One approach involves modification of rigid inorganic matrices such as sili ca. Initial work made it clear that di rect exposure of proteins to the sur face of small silica particles must be avoided, and could not be adequately controlled by addition of modifiers such as carbowax. The obvious ap proach then is to cover the silica sur face with a carbohydrate-like bonded phase, in order to duplicate the suc cess of the classical polysaccharide supports. The strategy is that such a packing could then be derivatized fur ther as with the classical phases to lead to ion-exchange and hydrophobic packings which are pressure stable. Unfortunately, many potential

ANALYTICAL CHEMISTRY, VOL. 5 0 . NO. 12, OCTOBER

1978

problems confront this approach. Un reacted and accessible silanol groups present a problem, particularly with basic proteins; thus, maximum cover age is necessary. In addition, there is a question concerning the stability of hydrophilic bonded phases in aqueous salt media. Nevertheless, some idea of the potential of high-speed LC for protein analyses can be seen in Figure 6 in the 4-min separation and quanti tation of three isoenzymes of CPK on a silica packing containing a glycerollike bonded phase with DEAE groups (89). A postcolumn, coupled-enzyme reaction was employed for detection in this case. Analysis of these isoenzymes is important in the diagnosis of myo cardial infarction. A second strategy is to develop pressure-stable hydrophilic organic gels with small dp's. Much progress is being made in this area, particularly among researchers in Japan. For ex ample, at the recent Japanese-Ameri can LC meeting in Boulder, Colo., Rokushika, Ohkawa, and Hatano showed some recent work on a ToyaSoda TSK-GEL 3000SW (115). GPC sepa rations of a number of proteins were illustrated with good recoveries and high plate counts (e.g., a-chymotrysinogen = 20 000 plates/m at low flow rates). The TSK SW and PW gels are not yet available in the U.S., but plans are underway to market them in this country. At this juncture it is difficult to pre dict which approach (i.e., bonded phase on silica gel or organic gel) will be the more successful. Alternatively, an approach involving some other rigid (or semirigid) porous matrix may be the packing of choice. What is clear, however, is that the availabilty of high-performance packings would revolutionize the chromatographic separations of proteins. Acknowledgment

The authors are appreciative of the review of this paper by L. R. Snyder and J. J. Kirkland. References

(1) J. J. DeStefano and J. J. Kirkland, Anal. Chem., 47,1103A, 1193A (1975). (2) P. T. Kissinger, ibid., 49,447A (1977). (3) E. Grushka and E. J. Kikta, Jr., ibid., ρ 1004A. (4) L. R. Snyder and J. J. Kirkland, "In troduction to Modern Liquid Chroma tography", Interscience, New York, N.Y., 1974. (5) "Practical High Performance Liquid Chromatography", C. F. Simpson, Ed., Heyden Press, New York, N.Y., 1976. (6) P. A. Bristow, "LC in Practice", HETP Publ., 10 Langley Dr., Handforth, Wilraslow, Cheshire. UK, 1976. (7) R.P.W. Scott, "Contemporary Liquid Chromatography", in A. Weissberger, Ed., "Techniques in Chemistry", Vol 11, Interscience, New York, N.Y., 1976. (8) H. Colin and G. Guiochon, J. Chromatogr., 141, 289 (1977).

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Barry L. Karger is professor of chemistry and director of the Institute of Chemical Analysis at Northeastern University. During 1972 he was visiting professor at Ecole Polytechnique, Paris, and the University of Saarbrucken, Germany. Dr. Karger has published extensively in the field of separation science, with recent emphasis on high-performance liquid chromatography. He was awarded a Sloan Fellowship (1971-1973), the Steven Dal Nogare Prize in Chromatography (1975), and the Northeastern University Lectureship (1976). He is on the editorial board of a number of journals including ANALYTICAL CHEMISTRY. Dr. Karger is a member of the ACS, Analytical Division of the ACS (currently councilor of the division), AAAS, Chromatography Discussion Group, London, and the New York Academy of Sciences. ANALYTICAL CHEMISTRY,

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HPLC research supported by NIH under grant GM 15847 and NSF under grant CHE-77-27900.

Roger W. Giese received his BS degree in chemistry at Hamline University in 1961 and his PhD in synthetic organic chemistry as a Woodrow Wilson Fellow at Massachusetts Institute of Technology in 1965. He joined the Department of Medicinal Chemistry as assistant professor and director of the MS Program in Clinical Chemistry at Northeastern University in 1974, and became a faculty fellow in the Institute of Chemical Analysis in 1975. He was promoted to associate professor and became a diplomate of the American Board of Clinical Chemistry in 1977. His research interests include the application of organic, chromatographic, and immunochemical methodology to clinical analysis. Dr. Giese is a member of the ACS, AAAS, the American Association for Clinical Chemistry, the Chemical Society, and the Clinical Radioassay Society. .. 50, NO. 12, OCTOBER 1978 · 1073 A

Reversed Phase Liquid Chromatography and Its Application

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