- Email: [email protected]
LIQUID CHROMATOGRAPHY | Biotechnology Applications
258 LIQUID CHROMATOGRAPHY / Biotechnology Applications
amino acids could not be resolved on the MIP because of the loose fit into the cavity.
See also: Derivatization of Analytes. Fluorescence: Derivatization. Liquid Chromatography: Amino Acids. Pharmaceutical Applications.
Chiral Mobile Phase Additives
There are no fundamental differences between the techniques using CSPs and chiral mobile phase additives. This means that all chiral selectors covalently bound to or coated on supports can be used for the enantioseparation of amino acids by addition to the mobile phase. The most frequently used additives in chiral ligand-exchange chromatography are Cu(II) complexes of chiral ligands such as D- or L-proline, esters of L-proline or L-arginine, N2-octyl-(S)-phenylalaninamide, or N-(p-toluenesulfonyl)-L-phenylalanine. Underivatized amino acids, amino acid amides, amino acid esters, and dansyl amino acids were separated using achiral C18 or ion-exchange columns. Figure 6 shows the effect of the chiral ligand on the enantioseparation of underivatized amino acids. When a Cu(II) complex of L-proline ligand was used as the mobile phase additive, the L-enantiomers of amino acids eluted before the corresponding D-enantiomers (Figure 6A). When the antipode was used as the ligand, the elution order was reversed (Figure 6B). Furthermore, no enantiomeric resolution of amino acids was observed with a racemic Cu(II)–proline complex (Figure 6C). CDs or derivatized CDs were used for the enantioseparation of dansyl- or t-butyloxycarbonylamino acids as the chiral mobile phase additive. One macrocyclic antibiotic, LY33328, was used for the enantioseparation of nine dansyl amino acids.
Further Reading Allenmark S (1991) Chromatographic Enantioseparation: Methods and Applications, 2nd edn. Chichester: Ellis Horwood. Bhushan R and Joshi S (1993) Resolution of enantiomers of amino acids by HPLC. Biomedical Chromatography 7: 235–250. Hamase K, Morikawa A, and Zaitsu K (2002) D-Amino acids in mammals and their diagnostic value. Journal of Chromatography B 781: 73–91. Krstulovic AM (ed.) (1989) Chiral Separations by HPLC: Applications to Pharmaceutical Compounds. Chichester: Ellis Horwood. Lough WJ (ed.) (1989) Chiral Liquid Chromatography. London: Blackie. Stevenson D and Wilson ID (eds.) (1988) Chiral Separations. New York: Plenum. Taylor DR and Maher K (1992) Chiral separations by high-performance liquid chromatography. Journal of Chromatographic Science 30: 67–85. Toyo’oka T (1996) Recent progress in liquid chromatographic enantioseparation based upon diastereomer formation with fluorescent chiral derivatization reagents. Biomedical Chromatography 10: 265–277. Wainer IW (1993) Drug Stereochemistry: Analytical Methods and Pharmacology, 2nd edn. New York: Dekker. Zief M and Crane LJ (eds.) (1988) Chromatographic Chiral Separations. New York: Dekker.
Biotechnology Applications R Freitag, Ecole Polytechnic Federal Lausanne, Lausanne, Switzerland & 2005, Elsevier Ltd. All Rights Reserved.
Introduction Biotechnology, i.e., the development and application of expertise from the disciplines of biology, chemistry, and engineering, represents a rapidly growing modern industry, which is increasingly being considered an important driving factor in many of today’s economies. The spectrum of biotechnological products covers a wide range from simple substances such as ethanol and citric acid through antibiotics and vaccines to the most advanced ‘biopharmaceuticals’
such as recombinant proteins, antibodies, or DNAbased therapeutics. Concomitantly a wide range of production organisms and conditions are used by the industry, while the application spectrum of ‘biotechnology’ covers areas as diverse as agriculture, food science, general industry, and, perhaps most importantly, medicine. However, there are certain common motifs in analytical biotechnology, which is mainly focused on process monitoring and control on the one side and quality control (identity, purity, stability) of the final product on the other. Monitoring the downstream process, i.e., product isolation and purification, then combines aspects of both areas. Analytical biotechnology has to operate within rather strict boundaries. In particular, the most precious
LIQUID CHROMATOGRAPHY / Biotechnology Applications 259
bioproducts (recombinant), proteins and DNA molecules, are not susceptible to analysis by methods such as gas chromatography and other standard high-resolution methods of analytical chemistry. In this context, liquid chromatography (LC) – together with the various electrophoretic methods – has been and will continue to be one of the major analytical techniques utilized for such work.
LC The most significant advantages of LC as an analytical technique in general are the diversity of parameters that can be used to achieve separation. For applications in analytical biotechnology, the most important of these parameters are size (gel permeation chromatography (GPC), size exclusion chromatography), charge (cation/anion exchange chromatography (IEC)), hydrophobicity/polarity (reversed-phase chromatography (RPC), hydrophobic interaction chromatography (HIC), normal-phase chromatography), and finally certain ‘biospecific’ interactions (affinity chromatography). By employing several of these techniques in series (multidimensional chromatography, e.g. IEC–HIC), highly orthogonal separation schemes can be drawn up that allow resolving even the most complex sample mixtures, e.g. for applications such as in ‘proteomics’. An alternative to such 2D LC techniques is coupling a high-resolution LC technique such as RPC with a second analytical technique such as matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry, i.e., creating a so-called hyphenated technique. Especially when micro- or nano-LC techniques are used, hyphenated techniques have advantages in terms of sample size, speed, detection limit, and solvent consumption. The most recent developments, true microanalytical LC-systems, that work with capillary columns or perform the entire analysis on a microfluidic chip (total analytical system). In such systems the content of a single cell becomes in principle accessible to a detailed analysis. Separation Mechanisms
The various LC techniques available today to the analytical chemist are discussed in detail in other chapters and sections of this encyclopedia. Below therefore only the methods and features relevant to analytical biotechnology are briefly discussed. As the typical separation problems of analytical biotechnology are similar to those encountered in other areas of (bio)analytical chemistry, commercially available standard stationary phases and prepacked columns are used normally. The stationary phases are
normally porous or pellicular (solid), spherical particles with a diameter of 2 or 10 mm and a very narrow particle size distribution (monosized beads) to ensure a high separation efficiency and minimum column back pressure simultaneously. Even complex separations can be carried out within minutes using such columns. In addition, so-called monolithic materials (UNOTM, BioRad USA, CIMTM-disks, BIA d.d.o, Slovenia) are being used increasingly, where the entire column consists of a single porous polymer block. Due to the improved mass transfer properties, such columns can be operated at extremely high flow rates without losing their efficiency. RPC Reversed-phase high-performance liquid chromatography (RP-HPLC) is perhaps the most important LC method in analytical biotechnology. The stationary phase is usually a silica composite bearing hydrophobic hydrocarbon groups (C2, C4, C8, or most often C18). Normally hydro-organic mobile phases (acetonitrile/water, methanol/water) are used, although variants using nonaqueous mobile phases have been reported. Elution is achieved by decreasing the polarity, i.e., increasing the organic content of the mobile phase. In the case of charged analytes, ionpairing reagents such as trifluoracetic acid (TFA) are added in small amounts to the mobile phase in order to reduce the nonspecific interaction between the analytes’ charges with residual accessible silanol groups on the silica surface. Due to their hydrophilicity, many biomolecules elute early in the gradient. While C18-phases are still used in the majority of analytical applications, less hydrophobic phases bearing shorter hydrocarbon groups are gradually becoming available. Another consequence of the low hydrophobicity of typical biomolecules is the need for compatibility of the stationary phase with mobile phases of low organic solvent content. While most of the earlier C18-phases, for example, could not be used with a mobile phase consisting of 100% water (irreversible collapse of the C18-brush structure), today several column types are available that can be used with gradients ranging from 0 to 100% of an organic modifier in the mobile phase. In analytical biotechnology, RP-HPLC is used for process monitoring, especially of media compounds such as certain amino acids but also for analysis of products such as antibiotics, enzymes, and other proteins. In addition, RP-HPLC constitutes the most important method for establishing the identity of many biotech products. In the case of recombinant proteins this may include the retention time of the product itself, the analysis of the amino acid composition, the analysis of the peptide map, as well
260 LIQUID CHROMATOGRAPHY / Biotechnology Applications
those of N-terminal variants, glycovariants, disulfide isomers, and proteolytic clips. Due to the volatility of its typical mobile phases, RP-HPLC has also become the method of choice for LC–mass spectrometry (MS) coupling. Ion-exchange chromatography Ion exchangers can be divided into cation exchangers and anion exchangers, with a further division into strong and weak ones according to the functional groups. The choice between a cation and anion exchanger will depend upon the net charge of the analyte under the conditions used – anion exchange for negatively charged analytes and cation exchange for positively charged ones. Whether to use a strong or weak exchanger is a more difficult decision. The charge density of a strong ion exchanger is essentially independent of pH, but for a weak ion exchanger it is dependent upon the pKa of the stationary phase. Ion exchange chromatography is carried out normally with an aqueous mobile phase (buffer) and tends to preserve biological activity to a high extent. Elution in IEC is accomplished by changing the pH or – most commonly – by increasing the ionic strength of the mobile phase. In analytical biotechnology IEC is used for process monitoring (amino acids, sugars, certain media compounds and products) and for quality control purposes, for example, for determination of glycosylation variants or detection of deamidated product molecules (proteins). In addition, IEC type stationary phases are also used in many sample preparation schemes for rapid enrichment of the target molecule-containing fraction prior to further analysis. While being a most powerful and widely used preparative separation technique, analytical IEC has the disadvantage of being difficult to couple to MS due to the need for a high salt elution buffer. For the same reason, miniaturization is difficult since high salt buffers are intrinsically unsuited for electrochromatographic applications, i.e., chromatographic separation, where the electro-osmotic flow is used to drive the mobile phase. Pressure-driven m-LC is possible in the IEC mode but is more difficult to perform. Gel filtration (size exclusion) Gel filtration chromatography (also called size exclusion chromatography) employs porous beads with a defined pore size distribution as the stationary phase. Small molecules can enter the entire intraparticular pore space and hence elute last, whereas large molecules are excluded from all pores and hence elute first. Molecules of intermediate size are found that can enter a certain
portion of the intraparticle pores. For these molecules the column residence time is a direct function of their size and they can hence be separated according to this parameter. Retentive (or repulsive) interactions between the analytes and the stationary phases have to be avoided, and the chromatographic conditions (mobile phase composition, stationary phase material) have to be chosen accordingly. In analytical biotechnology, GPC is used mainly for quality control, e.g., to differentiate between dimers/product aggregates and the actual product during purity and stability testing. Affinity chromatography In affinity chromatography the high specificity associated with the formation of a complex by two biological molecules is exploited. Ligands with specific binding sites, e.g., the antibody–antigen system, or group-specific molecules, e.g., lectins for the isolation of glycoproteins or Protein A for certain antibodies, can be used. Increasingly, affinity ligands are coming out of specific screening programs, based on, for example, combinatorial chemistry or biological systems such as phage display. The affinity ligand is linked covalently to an inert matrix. A spacer arm may be required, particularly for small molecules for decreasing matrix effects or steric hindrance to the interaction with the target molecules. Due to its high selectivity, affinity chromatography is a very important preparative technique in the biotechnological downstream process. In analytical biotechnology it is used in some cases to verify biological activity but mostly as a first capturing step for enriching a highly diluted product from the production stream prior to analysis using other techniques or for specific production rate monitoring during fermentation. The fact that often product variants (dimers, fragments, deamidated molecules, glycovariants) bind equally well to the affinity column may present a problem. Recently the coupling of affinity chromatography to MS has become more feasible, with in-line dialysis modules becoming available.
Amino Acids Determination of the amount and distribution of amino acids is required for a variety of bioanalytical questions. In bioprocess monitoring, the amount of certain free amino acids has to be monitored in order to avoid media depletion. In the case of a protein or a protein hydrolysate, analysis or verification of the amino acid composition may be a first step toward identification or characterization. The early work on amino acid analysis has been dominated by Stein and Moore, who in 1972 were awarded the Nobel prize
LIQUID CHROMATOGRAPHY / Biotechnology Applications 261
in chemistry for their work. The first dedicated amino acid analyzer was introduced by D.H. Spackman in 1950. Even today, the analysis of free amino acids is still done very much as proposed by these researchers, although considerable progress has been made in terms of the detection limit and the required sample volume. Analysis of free amino acids has to face two major difficulties. First, amino acids are small molecules that cover a wide range of polarity; they are therefore inherently difficult to handle for most LC techniques except IEC. Second, save for the aromatic amino acids, they possess almost no ultraviolet (UV) or fluorescence activity and can therefore only be detected using indirect methods, i.e., after labeling with a suitable dye. The introduction of a label prior to chromatography (precolumn labeling) will change the chromatographic behavior of the molecules and make them, for example, suitable for analysis using RP-HPLC. Compared with IEC, the separation will become faster and the resolution should improve in such cases. Today, analysis of free amino acids is normally carried out using IEC together with postcolumn derivatization for detection or by RP-HPLC together
with precolumn derivatization (Figure 1). The detection limit of precolumn techniques tends to be one order of magnitude lower (o1 pmol). At such levels, interference by impurities (microbial growth in column and/or buffer) may become a severe problem. The majority of the commercially available amino acid analyzers use IEC resins for the separation and ninhydrin as the postcolumn chromogenic reagent, although ortho-phthalaldehyde is gaining in popularity. Derivatization Reactions
Amino acids are derivatized in order to enable their detection by standard chromatographic detectors. Even today the perfect label has not been found. Such an ideal label should be easy to introduce in a defined manner (only one reaction product per amino acid) and should result in a stable and highly active (UVabsorbance, fluorescent activity) amino acid derivative, and it should be universal and react with all possible amino acids of interest. Dyes for postcolumn derivatization Ninhydrin has been the standard reagent for detecting amino acids
ILE
1.0 CA
LEU Internal standard
ASN
GLU
ALA TYR HYL
MSO
SER
MET VAL
GLN
TRP
ASP
50
LYS
ARG PHE
THR GLY
Concentration of methanol (% v / v)
CMC
Fluorescence (µA)
100
HIS
IMP 0
0.0 0
10
20
30
40
50
t (min) Figure 1 Separation of a physiological mixture of amino acids. (Reproduced with permission from Agnes H (ed.) (1985) HighPerformance Liquid Chromatography in Biochemistry. Wiley-VCH, p. 156.)
262 LIQUID CHROMATOGRAPHY / Biotechnology Applications
for over 50 years. However, not all amino acids react equally well with ninhydrin, and the detection limit is B50 pmol (B6 ng) of amino acid derivative. The product of the reaction of ortho-phthaldialdehyde (OPA) with amino acids in the presence of thiol will fluoresce with excitation at 340 nm and emission at 450 nm. The reaction is fast (1–3 minutes), however, the OPA-derivatives are not stable. Secondary amino acids do not react and must first be converted into primary amino acids (oxidation with hypochlorite or chloramines-T (N-chloro-p-toluenesulfonamide). The detection limit in the case of postcolumn derivatization with OPA is B10 pmol (1.2 ng). Fluorescein has also been proposed for postcolumn derivatization of free amino acids but is not widely used due to the many problems associated with its application.
Conversion of amino acids to produce a diastereoisomeric peptide by reaction with an N-carboxyanhydride, e.g., L-Phe-N-carboxy-anhydride, has been used for determination of optical purity using RPC. Alternatively, for analysis of amino acid enantiomers without derivatization, two options are available: (1) chiral mobile phases such as the N,N-di-n-propyl-L-alanine–copper(II) complex can be used with reversed-phase columns, and (2) stationary phases with a covalently bound ligand capable of stereo recognition can be used. Such ligands include cyclodextrins, albumins, glycoproteins, and copper(II) complexes.
Dyes for precolumn derivatization The Edman reagent (phenylisothiocyanate) can be used to form thiohydantoin derivatives of primary and secondary amino acids, which can be separated using RPC (detection UV 254 nm). The detection limit is B1 pmol (B0.1 ng). OPA is also used for the precolumn derivatization. Detection limits (fluorescence) down to the femtomole level are possible. Some other dyes are of limited importance in precolumn derivatization. 4-(Dimethylamino)azobenzene-40 -sulfonyl chloride reacts with amino acids to produce a derivative that is stable at room temperature and can be detected at the 3–5 pmol level using detection at 436 nm. 4-(Dimethylamino)azobenzene-40 -isothiocyanate in combination with RPC has a detection limit of B5 pmol with detection at 436 nm. Fluorenylmethoxycarbonyl chloride forms derivatives of all amino acids, including proline. UV detection is possible at 260 nm, and fluorescence detection (excitation 266 nm, emission 305 nm) is also possible, the detection limit being B50 fmol in that case. 5-(Dimethylamino)naphthalene-1-sulfonyl chloride also reacts with both primary and secondary amines. The derivatives can be detected using UV at 254 nm or using fluorescence (excitation 385 nm, emission 460 nm). The sensitivity is in the low picomole range. Due to some problems with the specificity and the completeness of the reaction, the dye is rarely used.
The analysis of peptides and proteins naturally has some analogies with that of their main building blocks, amino acids (Figure 3). The analysis of proteomes, i.e., the entire gene product spectrum expressed by a given organism (cell) at a given time, is a very recent development in protein analysis. Proteome analysis is largely dominated by 2D gel electrophoresis (orthogonal separation of the protein mixture according to isoelectric point and mass) as a literature survey will show that still well over 90% of the pertinent separations are carried out using this method. However, the application of orthogonal LC–LC and LC–MS techniques for this purpose is gaining ground. For analysis of proteins and (synthetic) peptides, on the other hand, LC techniques are the method of choice (Figure 4). The problems encountered in analysis of free amino acids, namely the lack of an intrinsic detection method and the wide polarity range, are much less significant in the case of proteins/peptides. Since most of these molecules contain at least one aromatic amino acid and several peptide bonds, their UV detection at 280 nm and 214 nm, respectively, is straightforward. Since most peptides/ proteins are made for a mixture of polar and nonpolar amino acid residues, their separation using RPHPLC in a gradient of increasing organic solvent is the most commonly used approach. The resolution of RP-HPLC is high enough to show clear differences between a protein and its variants (reduced, deamidated form, etc.) and also between the correct product of a peptide synthesis and peptides where one or several addition steps have failed. A second important method for protein characterization and identification is the peptide map. For this purpose the protein is digested by a protease such as trypsin (tryptic map) or chymotrypsin, and the resulting peptide mix is analyzed using RP-HPLC. Even single
Enantiomeric Separations
The need to resolve the D- and L-enantiomers of amino acids has grown in recent years as it has been recognized that they differ in biological and physicochemical properties. Amino acid enantiomers cannot be resolved in achiral systems; it is therefore necessary to have a second chiral center either in the chromatographic system or to create one by derivatization in the molecule to be separated (Figure 2).
Peptides, Proteins, ‘Proteomics’
LIQUID CHROMATOGRAPHY / Biotechnology Applications 263
1
2
4
3
7 8
13, 14
6 5
9
10 11
12
15 16
0
20
40
60
80
Retent. time (min) Figure 2 Analysis of eight amino acid racemates. Chromatography conditions: column MCI GEL CRS10W, eluent 0.5 mmol l 1 copper (II) sulfate, flow rate 1.0 ml min 1, detection 254 nm, peaks 1 D-Ala, 2 L-Ala, 3 D-Pro, 4 D-Val, 5 L-Pro, 6 L-Val, 7 D-Leu, 8 D-Nle, 9 D-Tyr, 10 L-Leu, 11 D-Eth, 12 L-Tyr, 13 L-Nle, 14 D-Phe, 15 L-Eth, 16 L-Phe. (Reproduced with permission from Weston A and Brown Ph (1997) HPLC and CE – Principles and Practice. Academic Press, p. 61; & Elsevier.)
amino acid deviations can normally be detected as differences in the peak pattern given by the protein product and the variants. Peptide mapping is in addition very important for an indirect evaluation of the glycosylation pattern as the glycoform of a given peptide will show significant differences in the retention behavior compared with the nonglycosylated form. Finally, in combination with MS, the identity of the separated peptides can be determined and compared with the theoretical one in the case of proteins with known primary structure.
Carbohydrates Carbohydrate analysis has been slow to develop in comparison with that of other biologically important molecules. However, the more recent developments in biotechnology and renewable resources of raw materials have promoted interest in this diverse class
of compounds. Indeed, the term ‘carbohydrate’ encompasses a wide range of compounds from the simple building blocks (monosaccharides) to carbohydrate polymers, oligosaccharides, and polysaccharides and to various carbohydrate-containing species including glycoproteins, proteoglycans, nucleic acids, glycolipids, and antibiotics. Monosaccharides
There is a great variety in structure as well as chemical and physical properties of monosaccharides. There is no single method that is applicable to the qualitative and quantitative analysis of all monosaccharides; instead, the method must be chosen according to the chemical and physical characteristics of the solutes of interest. Borate complexes of monosaccharides can be separated using strong (quaternary amine, Q-type) anion-exchange columns. Alternatively, if the monosaccharide is acidic
264 LIQUID CHROMATOGRAPHY / Biotechnology Applications
or negatively charged at high pH, anion exchangers can be used directly. Cation-exchange chromatography is normally used for analysis of amino sugars and – using calcium as counterion – alditols and monosaccharides. Oligosaccharides
Oligosaccharides are traditionally defined as polymers of monosaccharides containing from two to ten
A
Absorbance at 215 nm
0.10
B
C
0.05
0.0 0
3
0
4 0 Time (min)
6
Figure 3 Determination of protein purity. Chromatographic conditions: column Hy-Tach micropellicular C-18 silica, eluent A 0.1% TFA in water, eluent B 95% acetonitrile in water containing 0.1% TFA, flow rate 2.0 ml min 1, sample A 40 mg carbonic anhydrase (gradient 15 to 55% B in 3 min), B 40 mg L-asparaginase (gradient 30 to 40% B in 4 Lmin), C 40 mg myoglobin (gradient 23 to 45% B in 6 min). (Reproduced with permission from Horvath C and Nikelly JG (eds.) (1990) Analytical Biotechnology, ACS Symposium Series 434; & American Chemical Society.)
residues, although this range is often extended to 25 as naturally occurring polysaccharides rarely contain less than 25 repeat units. Anion-exchange resins with acetate, hydroxyl, or chloride counterions are usually used for oligosaccharide analysis. For a homologous series, the elution increases with increasing chain length. In an attempt to overcome the problems of interconversion of the terminal reducing residues often encountered in anion-exchange chromatography of oligosaccharides, cation-exchange resins, in particular sulfonated poly(styrene–divinyl–benzene) materials, have been used for oligosaccharide separations, with water as the mobile phase. The effect of the counterion, e.g., lithium, barium, potassium, silver, or lead, is considerable as the separation mechanism is not purely cation exchange but also encompasses ligand exchange. Gel filtration chromatography is another very useful technique for analysis of oligosaccharides. By a judicious choice of the stationary phase, a resolution of up to DP 15 can be achieved. The use of RPC for oligosaccharide analysis, on the other hand, has in general been limited to smaller oligomers owing to problems of solubility in the typical hydro-organic mobile phases. Normal-phase chromatography using stationary phases with amino or cyano functionalities has been used extensively for oligosaccharide analysis, again with aqueous acetonitrile mobile phases. However, unlike in RPC, elution takes place in a gradient of increasing water content of the mobile phase. Higher oligomers or polysaccharide material may be bound irreversibly to the column or have excessively long elution times.
r t-PA reference standard arginine at position 275
Mutant form of r t-PA with Glutamic acid at position 275 0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
Time (min) Figure 4 Comparison of the tryptic map of a recombinant tissue plasminogen activator reference standard and a mutant form with a glutamic acid residue instead of the normal arginine residue at position 275. (Reproduced with permission from Weston A and Brown Ph (1997) HPLC and CE – Principles and Practice, Academic Press, p. 37; & Elsevier.)
LIQUID CHROMATOGRAPHY / Biotechnology Applications 265 High molecular mass
I
6
II
III IV
7 1 3 12 8 13 11 15 10 9 14
5
4
2 2 3
1
5
4 A 3
6 Time (h)
9
Figure 5 Gel filtration chromatography of starch-derived oligosaccharides on a Bio-Gel P2 column. Peak numbers refer to the degree of polymerization.
B
C
Polysaccharides
The number of liquid chromatographic methods available for analysis of polysaccharides is limited compared with those for monomers or oligomers. Polysaccharides comprise a diverse group of complex macromolecules that may range in molecular mass from several thousand to several million Daltons and may be composed of neutral, acidic, or basic monomers or mixtures thereof arranged in a variety of structures. As polysaccharides are polydisperse macromolecules, gel filtration is often the first liquid chromatographic method to be used for their characterization. As the separation mechanism is one based on solute size, columns can be calibrated with polysaccharides of known molecular masses, and estimates for unknowns can be made. IEC is used to assess the homogeneity of a polysaccharide, often in combination with gel filtration chromatography, to separate molecules of similar size on the basis of differences in their charges (Figure 5). Carbohydrate-Containing Macromolecules, Glycoproteins
The methods previously discussed for mono-, oligo-, and polysaccharides are also applicable to analysis of carbohydrate-containing macromolecules. For example, proteoglycans, which consist of a central core protein to which a number of glycosaminoglycan chains are attached, which carry a significant negative charge due to a high concentration of carboxyl and sulfate ester groups, can be separated using anion-exchange chromatography (Figure 6). Of increasing importance in medical biotechnology is analysis of the glycosylation pattern, i.e., the composition and structure of the oligosaccharide chains of glycoproteins. While the amino acid sequence is encoded by the genetic code and can hence be transferred by genetic engineering from one
D
E
12
14
16
18 20 time (min)
22
24
Figure 6 HPAEC–PAD mapping of N- and O-glycans of glycoproteins after release by hydrazinolysis. (Reproduced with permission from Animal cell technology: products from cells, cells as products in Proceedings of the 16th ESACT Meeting, April 25–29, Lugano, Switzerland ; & Kluwer Academic Publishers.)
species to the next, glycosylation is a post-translatorial event and hence shows pronounced differences from species to species and even between the different cell types of a given organism. At the same time glycosylation does have significant consequences for the biological activity as well as for the pharmacokinetics and -dynamics. A typical procedure in analysis of glycosylation patterns is comparison of the peptide map of the native glycoprotein and of that of the residual polypeptide structure after enzymatic release of the sugar residues together with a direct analysis of the released glycans using high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). Detection Methods
In general carbohydrates do not have a UV chromophore and do not fluoresce. Monosaccharides do absorb in the far UV, with the absorbance maxima at B188 nm. Detection is normally performed at wavelengths between 192 and 200 nm, where there is less residual noise in the detector signal, if the absorbance of the solvent permits this. The UV
266 LIQUID CHROMATOGRAPHY / Biotechnology Applications 1 2 8
5
10
7 3 4
13 6 9
11
12
automated versions of the spectrophotometric assays traditionally used for identification and quantification of carbohydrates. Methods of detection for total carbohydrate include the following: L-cysteine–sulphuric acid- and phenol–sulfuric acid-based assays for neutral carbohydrates, the carbazole assay or Warren assay for acid carbohydrates, the Elson Morgan assay for basic carbohydrates, and the 3,5-dinitrosalicylic acid assay for reducing groups.
Organic Acids
24 Time (min)
48
Figure 7 Ion exclusion isocratic separation of organic acids. Peaks: 1 chloride, 2 oxalate, 3 pyruvate, 4 tartrate, 5 malonate, 6 lactate, 7 malate, 8 acetate, 9 isocitrate, 10 citrate, 11 b-hydroxyn-butyrate, 12 succinate, 13 proprionate. (Reproduced with permission from Dionex Product Selection Guide 1991.)
response is dependent upon the carbonyl group and will differ for the individual monosaccharides, fructose and glucose being the two extremes, with the response of fructose being six times that of glucose. Differential refractive index detection has often been used in the past; however, the method is not particularly sensitive and is only suitable for isocratic separations. Increasingly, PAD is being used as the universal method in the LC of mono- and oligosaccharides (Figure 7). The carbohydrate is oxidized at a gold electrode at a pH greater than 11, with the electrode surface being reduced back to gold using a negative potential sequence. In addition, carbohydrates can be derivatized to produce UV or fluorescent species to improve detection. As with the amino acids, both precolumn and postcolumn derivatization are used. Precolumn derivatization influences the chromatographic behavior of the compounds. For UV detection, phenylisocyanate derivatization can be used together with RP-HPLC or benzoyl chloride and 4-bromobenzoyl chloride derivatization together with normal-phase chromatography. When fluorescence detection is preferred, dansyl hydrazine and aminopyridine derivatives can be analyzed using RPC. Postcolumn derivatization is normally preferred for carbohydrate analysis as it is applicable to mono-, oligo-, and polysaccharides. It also improves the detection limit, and of course analysis selectivity, but without altering the intrinsic separation characteristics of the carbohydrates by introducing a group or groups that may dominate the separation process. The most common derivatization reactions are
Short-chain mono-, di-, and polycarboxylic acids are of significance to scientists studying amino acid degradation, metabolites of carbohydrate oxidation, and lipid breakdown. They can be indicators of human metabolic disorders and of importance in the biotechnology of food. LC is now the preferred method of analysis for these analytes irrespective of the sample origin – biological fluid, fermentation broth, or food. IEC, or perhaps more correctly ion-exclusion chromatography using sulfonated polymeric resins, has become the method of choice for separating organic acids. The stationary phase can withstand extremes of pH, and the separation of the organic acids is normally according to pKa values, but partition effects can also occur. Dilute acids are used as the mobile phase. Normally only isocratic mobile phases are required, with the addition of a small quantity of an organic modifier (usually acetonitrile) for analysis of nonpolar acids. In the case of weak organic acids, ion pairing agents can be used and the analysis can then also be achieved using RP-HPLC. However, this separation system is limited to certain organic acids, and the reproducibility can be poor. Detection can be achieved at nanogram or picogram levels, using electrochemical and conductivity detectors.
Monitoring Fermentations and Fermentation Products LC continues to be an important tool in fermentation monitoring. Using modern technology, the methods used are sufficiently fast and robust even for fermentation control, for example, to supervise nutrient addition and hence to maintain an optimum environment for the production organisms. Carbohydrates, amino acids, and the content of organic acids, all critical to either the production or the inhibition of production, can all be monitored as described above. In the production of wine, monitoring the pyruvic acid content during malo-lactic fermentation gives an
LIQUID CHROMATOGRAPHY / Clinical Applications 267
indication of the health of the bacteria, a pyruvate being an indirect measure of NADH, the reducing co-factor that stimulates bacterial growth. Malates and tartrates are also simultaneously monitored, being indicators of the maturity of the fermentation process. Lactose fermentation can yield lactic, acetic, pyruvic, formic, propionic, and butyric acids, which are indicators of the flavor and stability of dairy products. All these acids can be determined in one LC-analysis run. In the food industry LC is frequently used to determine the quality of the finished product. For example, it has been shown that the flavor quality of grape juice is directly linked to the amount and spectrum of organic acids present. Likewise, in the production of sour cream by fermentation, the quality of the finished product quality is related directly to the organic acid profile. Last, but not least and as pointed out above, LC techniques are also crucial in determining the quality of recombinant protein products. See also: Carbohydrates: Overview; Sugars – Chromatographic Methods. Chromatography: Overview; Principles. Derivatization of Analytes. Electrophoresis: Two-Dimensional Gels; Proteins. Fluorescence: Derivatization; Fluorescence Labeling. Ion Exchange: Overview; Principles; Isolation of Biopolymers. Liquid Chromatography: Overview; Principles; Column Technology; Mobile Phase Selection; Normal Phase; Reversed Phase; Size Exclusion; Chiral; Affinity Chromatography; Multidimensional; Instrumentation; Liquid Chromatography–Mass Spectrometry; Amino Acids; Clinical Applications. Micro Total Analytical Systems. Peptides. Pharmaceutical Analysis: Overview. Process Analysis: Chromatography; Bioprocess Analysis.
Proteins: Traditional Methods of Sequence Determination. Proteomics.
Further Reading Chaplin MF (1986) Monosaccharides. In: Chaplin MF and Kennedy JF (eds.) Carbohydrate Analysis – A Practical Approach, pp. 37–69. Oxford IRL Press. El Rassi Z (ed.) (1995) Carbohydrate Analysis. Journal of Chromatography Library, vol. 58. Elsevier. Heftmann E (ed.) (1991) Chromatography, 5th edn., Journal of Chromatography Library, vol. 51. Elsevier. Horvath C and Nikelly JG (eds.) (1990) Analytical Biotechnology. ACS Symposium Series 434. Washington, DC: American Chemical Society. Kastner M (ed.) (2000) Protein Liquid Chromatography. Journal of Chromatography Library, vol. 67. Elsevier. Keller R, Lottspeich F, Meaer HE (eds.) (1999) Microcharacterization of Proteins. Wiley–VCH. Lloyd LL, Warner FP, and Kennedy JF (1990) The application of HPLC in biotechnology. In: Biotechnology International, pp. 221–226. London: Century Press. Schwarzenbach R (1982) High-performance liquid chromatography of carboxylic acids. Journal of Chromatography 251: 339–358. Sofer G and Hagel L (1997) Handbook of Process Chromatography – A Guide to Optimization, Scale-up and Validation. Academic Press. Tukova J (ed.) (1993) Bioaffinity Chromatography, 2nd completely revised edn., Journal of Chromatography Library, vol. 55. Elsevier. Weston A and Brown Ph (1997) HPLC and CE – Principles and Practice. Academic Press. White CA and Kennedy JF (1986) Oligosaccharides. In: Chaplin MF and Kennedy JF (eds.) Carbohydrate Analysis – A Practical Approach, pp. 37–69. Oxford IRL Press.
Clinical Applications R A Sherwood, King’s College Hospital, London, UK & 2005, Elsevier Ltd. All Rights Reserved. This article is a revision of the previous-edition article by R A Sherwood and B F Rocks, pp. 2677–2685, & 1995, Elsevier Ltd.
Introduction The diagnosis and monitoring of many disease states relies on the detection and quantitation of a particular compound(s) or metabolite(s) in body fluids, usually blood or urine. A wide variety of techniques are used in the clinical chemistry laboratory with spectrophotometric and immunoassay methods on automated analyzers measuring single compounds
predominating. In some instances a family of compounds may need to be measured and it is in these situations where the chromatographic techniques are important to avoid the necessity for a separate assay for each component of interest in the mixture. Initially chromatography was carried out using paper or slab gels. The use of columns to contain the stationary phase came more recently with the advent of high-performance liquid chromatography (HPLC). Typical applications of HPLC in clinical chemistry include: 1. Biogenic amines (a) Catecholamines and metabolites (b) Serotonin and metabolites
amino acids could not be resolved on the MIP because of the loose fit into the cavity.
See also: Derivatization of Analytes. Fluorescence: Derivatization. Liquid Chromatography: Amino Acids. Pharmaceutical Applications.
Chiral Mobile Phase Additives
There are no fundamental differences between the techniques using CSPs and chiral mobile phase additives. This means that all chiral selectors covalently bound to or coated on supports can be used for the enantioseparation of amino acids by addition to the mobile phase. The most frequently used additives in chiral ligand-exchange chromatography are Cu(II) complexes of chiral ligands such as D- or L-proline, esters of L-proline or L-arginine, N2-octyl-(S)-phenylalaninamide, or N-(p-toluenesulfonyl)-L-phenylalanine. Underivatized amino acids, amino acid amides, amino acid esters, and dansyl amino acids were separated using achiral C18 or ion-exchange columns. Figure 6 shows the effect of the chiral ligand on the enantioseparation of underivatized amino acids. When a Cu(II) complex of L-proline ligand was used as the mobile phase additive, the L-enantiomers of amino acids eluted before the corresponding D-enantiomers (Figure 6A). When the antipode was used as the ligand, the elution order was reversed (Figure 6B). Furthermore, no enantiomeric resolution of amino acids was observed with a racemic Cu(II)–proline complex (Figure 6C). CDs or derivatized CDs were used for the enantioseparation of dansyl- or t-butyloxycarbonylamino acids as the chiral mobile phase additive. One macrocyclic antibiotic, LY33328, was used for the enantioseparation of nine dansyl amino acids.
Further Reading Allenmark S (1991) Chromatographic Enantioseparation: Methods and Applications, 2nd edn. Chichester: Ellis Horwood. Bhushan R and Joshi S (1993) Resolution of enantiomers of amino acids by HPLC. Biomedical Chromatography 7: 235–250. Hamase K, Morikawa A, and Zaitsu K (2002) D-Amino acids in mammals and their diagnostic value. Journal of Chromatography B 781: 73–91. Krstulovic AM (ed.) (1989) Chiral Separations by HPLC: Applications to Pharmaceutical Compounds. Chichester: Ellis Horwood. Lough WJ (ed.) (1989) Chiral Liquid Chromatography. London: Blackie. Stevenson D and Wilson ID (eds.) (1988) Chiral Separations. New York: Plenum. Taylor DR and Maher K (1992) Chiral separations by high-performance liquid chromatography. Journal of Chromatographic Science 30: 67–85. Toyo’oka T (1996) Recent progress in liquid chromatographic enantioseparation based upon diastereomer formation with fluorescent chiral derivatization reagents. Biomedical Chromatography 10: 265–277. Wainer IW (1993) Drug Stereochemistry: Analytical Methods and Pharmacology, 2nd edn. New York: Dekker. Zief M and Crane LJ (eds.) (1988) Chromatographic Chiral Separations. New York: Dekker.
Biotechnology Applications R Freitag, Ecole Polytechnic Federal Lausanne, Lausanne, Switzerland & 2005, Elsevier Ltd. All Rights Reserved.
Introduction Biotechnology, i.e., the development and application of expertise from the disciplines of biology, chemistry, and engineering, represents a rapidly growing modern industry, which is increasingly being considered an important driving factor in many of today’s economies. The spectrum of biotechnological products covers a wide range from simple substances such as ethanol and citric acid through antibiotics and vaccines to the most advanced ‘biopharmaceuticals’
such as recombinant proteins, antibodies, or DNAbased therapeutics. Concomitantly a wide range of production organisms and conditions are used by the industry, while the application spectrum of ‘biotechnology’ covers areas as diverse as agriculture, food science, general industry, and, perhaps most importantly, medicine. However, there are certain common motifs in analytical biotechnology, which is mainly focused on process monitoring and control on the one side and quality control (identity, purity, stability) of the final product on the other. Monitoring the downstream process, i.e., product isolation and purification, then combines aspects of both areas. Analytical biotechnology has to operate within rather strict boundaries. In particular, the most precious
LIQUID CHROMATOGRAPHY / Biotechnology Applications 259
bioproducts (recombinant), proteins and DNA molecules, are not susceptible to analysis by methods such as gas chromatography and other standard high-resolution methods of analytical chemistry. In this context, liquid chromatography (LC) – together with the various electrophoretic methods – has been and will continue to be one of the major analytical techniques utilized for such work.
LC The most significant advantages of LC as an analytical technique in general are the diversity of parameters that can be used to achieve separation. For applications in analytical biotechnology, the most important of these parameters are size (gel permeation chromatography (GPC), size exclusion chromatography), charge (cation/anion exchange chromatography (IEC)), hydrophobicity/polarity (reversed-phase chromatography (RPC), hydrophobic interaction chromatography (HIC), normal-phase chromatography), and finally certain ‘biospecific’ interactions (affinity chromatography). By employing several of these techniques in series (multidimensional chromatography, e.g. IEC–HIC), highly orthogonal separation schemes can be drawn up that allow resolving even the most complex sample mixtures, e.g. for applications such as in ‘proteomics’. An alternative to such 2D LC techniques is coupling a high-resolution LC technique such as RPC with a second analytical technique such as matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry, i.e., creating a so-called hyphenated technique. Especially when micro- or nano-LC techniques are used, hyphenated techniques have advantages in terms of sample size, speed, detection limit, and solvent consumption. The most recent developments, true microanalytical LC-systems, that work with capillary columns or perform the entire analysis on a microfluidic chip (total analytical system). In such systems the content of a single cell becomes in principle accessible to a detailed analysis. Separation Mechanisms
The various LC techniques available today to the analytical chemist are discussed in detail in other chapters and sections of this encyclopedia. Below therefore only the methods and features relevant to analytical biotechnology are briefly discussed. As the typical separation problems of analytical biotechnology are similar to those encountered in other areas of (bio)analytical chemistry, commercially available standard stationary phases and prepacked columns are used normally. The stationary phases are
normally porous or pellicular (solid), spherical particles with a diameter of 2 or 10 mm and a very narrow particle size distribution (monosized beads) to ensure a high separation efficiency and minimum column back pressure simultaneously. Even complex separations can be carried out within minutes using such columns. In addition, so-called monolithic materials (UNOTM, BioRad USA, CIMTM-disks, BIA d.d.o, Slovenia) are being used increasingly, where the entire column consists of a single porous polymer block. Due to the improved mass transfer properties, such columns can be operated at extremely high flow rates without losing their efficiency. RPC Reversed-phase high-performance liquid chromatography (RP-HPLC) is perhaps the most important LC method in analytical biotechnology. The stationary phase is usually a silica composite bearing hydrophobic hydrocarbon groups (C2, C4, C8, or most often C18). Normally hydro-organic mobile phases (acetonitrile/water, methanol/water) are used, although variants using nonaqueous mobile phases have been reported. Elution is achieved by decreasing the polarity, i.e., increasing the organic content of the mobile phase. In the case of charged analytes, ionpairing reagents such as trifluoracetic acid (TFA) are added in small amounts to the mobile phase in order to reduce the nonspecific interaction between the analytes’ charges with residual accessible silanol groups on the silica surface. Due to their hydrophilicity, many biomolecules elute early in the gradient. While C18-phases are still used in the majority of analytical applications, less hydrophobic phases bearing shorter hydrocarbon groups are gradually becoming available. Another consequence of the low hydrophobicity of typical biomolecules is the need for compatibility of the stationary phase with mobile phases of low organic solvent content. While most of the earlier C18-phases, for example, could not be used with a mobile phase consisting of 100% water (irreversible collapse of the C18-brush structure), today several column types are available that can be used with gradients ranging from 0 to 100% of an organic modifier in the mobile phase. In analytical biotechnology, RP-HPLC is used for process monitoring, especially of media compounds such as certain amino acids but also for analysis of products such as antibiotics, enzymes, and other proteins. In addition, RP-HPLC constitutes the most important method for establishing the identity of many biotech products. In the case of recombinant proteins this may include the retention time of the product itself, the analysis of the amino acid composition, the analysis of the peptide map, as well
260 LIQUID CHROMATOGRAPHY / Biotechnology Applications
those of N-terminal variants, glycovariants, disulfide isomers, and proteolytic clips. Due to the volatility of its typical mobile phases, RP-HPLC has also become the method of choice for LC–mass spectrometry (MS) coupling. Ion-exchange chromatography Ion exchangers can be divided into cation exchangers and anion exchangers, with a further division into strong and weak ones according to the functional groups. The choice between a cation and anion exchanger will depend upon the net charge of the analyte under the conditions used – anion exchange for negatively charged analytes and cation exchange for positively charged ones. Whether to use a strong or weak exchanger is a more difficult decision. The charge density of a strong ion exchanger is essentially independent of pH, but for a weak ion exchanger it is dependent upon the pKa of the stationary phase. Ion exchange chromatography is carried out normally with an aqueous mobile phase (buffer) and tends to preserve biological activity to a high extent. Elution in IEC is accomplished by changing the pH or – most commonly – by increasing the ionic strength of the mobile phase. In analytical biotechnology IEC is used for process monitoring (amino acids, sugars, certain media compounds and products) and for quality control purposes, for example, for determination of glycosylation variants or detection of deamidated product molecules (proteins). In addition, IEC type stationary phases are also used in many sample preparation schemes for rapid enrichment of the target molecule-containing fraction prior to further analysis. While being a most powerful and widely used preparative separation technique, analytical IEC has the disadvantage of being difficult to couple to MS due to the need for a high salt elution buffer. For the same reason, miniaturization is difficult since high salt buffers are intrinsically unsuited for electrochromatographic applications, i.e., chromatographic separation, where the electro-osmotic flow is used to drive the mobile phase. Pressure-driven m-LC is possible in the IEC mode but is more difficult to perform. Gel filtration (size exclusion) Gel filtration chromatography (also called size exclusion chromatography) employs porous beads with a defined pore size distribution as the stationary phase. Small molecules can enter the entire intraparticular pore space and hence elute last, whereas large molecules are excluded from all pores and hence elute first. Molecules of intermediate size are found that can enter a certain
portion of the intraparticle pores. For these molecules the column residence time is a direct function of their size and they can hence be separated according to this parameter. Retentive (or repulsive) interactions between the analytes and the stationary phases have to be avoided, and the chromatographic conditions (mobile phase composition, stationary phase material) have to be chosen accordingly. In analytical biotechnology, GPC is used mainly for quality control, e.g., to differentiate between dimers/product aggregates and the actual product during purity and stability testing. Affinity chromatography In affinity chromatography the high specificity associated with the formation of a complex by two biological molecules is exploited. Ligands with specific binding sites, e.g., the antibody–antigen system, or group-specific molecules, e.g., lectins for the isolation of glycoproteins or Protein A for certain antibodies, can be used. Increasingly, affinity ligands are coming out of specific screening programs, based on, for example, combinatorial chemistry or biological systems such as phage display. The affinity ligand is linked covalently to an inert matrix. A spacer arm may be required, particularly for small molecules for decreasing matrix effects or steric hindrance to the interaction with the target molecules. Due to its high selectivity, affinity chromatography is a very important preparative technique in the biotechnological downstream process. In analytical biotechnology it is used in some cases to verify biological activity but mostly as a first capturing step for enriching a highly diluted product from the production stream prior to analysis using other techniques or for specific production rate monitoring during fermentation. The fact that often product variants (dimers, fragments, deamidated molecules, glycovariants) bind equally well to the affinity column may present a problem. Recently the coupling of affinity chromatography to MS has become more feasible, with in-line dialysis modules becoming available.
Amino Acids Determination of the amount and distribution of amino acids is required for a variety of bioanalytical questions. In bioprocess monitoring, the amount of certain free amino acids has to be monitored in order to avoid media depletion. In the case of a protein or a protein hydrolysate, analysis or verification of the amino acid composition may be a first step toward identification or characterization. The early work on amino acid analysis has been dominated by Stein and Moore, who in 1972 were awarded the Nobel prize
LIQUID CHROMATOGRAPHY / Biotechnology Applications 261
in chemistry for their work. The first dedicated amino acid analyzer was introduced by D.H. Spackman in 1950. Even today, the analysis of free amino acids is still done very much as proposed by these researchers, although considerable progress has been made in terms of the detection limit and the required sample volume. Analysis of free amino acids has to face two major difficulties. First, amino acids are small molecules that cover a wide range of polarity; they are therefore inherently difficult to handle for most LC techniques except IEC. Second, save for the aromatic amino acids, they possess almost no ultraviolet (UV) or fluorescence activity and can therefore only be detected using indirect methods, i.e., after labeling with a suitable dye. The introduction of a label prior to chromatography (precolumn labeling) will change the chromatographic behavior of the molecules and make them, for example, suitable for analysis using RP-HPLC. Compared with IEC, the separation will become faster and the resolution should improve in such cases. Today, analysis of free amino acids is normally carried out using IEC together with postcolumn derivatization for detection or by RP-HPLC together
with precolumn derivatization (Figure 1). The detection limit of precolumn techniques tends to be one order of magnitude lower (o1 pmol). At such levels, interference by impurities (microbial growth in column and/or buffer) may become a severe problem. The majority of the commercially available amino acid analyzers use IEC resins for the separation and ninhydrin as the postcolumn chromogenic reagent, although ortho-phthalaldehyde is gaining in popularity. Derivatization Reactions
Amino acids are derivatized in order to enable their detection by standard chromatographic detectors. Even today the perfect label has not been found. Such an ideal label should be easy to introduce in a defined manner (only one reaction product per amino acid) and should result in a stable and highly active (UVabsorbance, fluorescent activity) amino acid derivative, and it should be universal and react with all possible amino acids of interest. Dyes for postcolumn derivatization Ninhydrin has been the standard reagent for detecting amino acids
ILE
1.0 CA
LEU Internal standard
ASN
GLU
ALA TYR HYL
MSO
SER
MET VAL
GLN
TRP
ASP
50
LYS
ARG PHE
THR GLY
Concentration of methanol (% v / v)
CMC
Fluorescence (µA)
100
HIS
IMP 0
0.0 0
10
20
30
40
50
t (min) Figure 1 Separation of a physiological mixture of amino acids. (Reproduced with permission from Agnes H (ed.) (1985) HighPerformance Liquid Chromatography in Biochemistry. Wiley-VCH, p. 156.)
262 LIQUID CHROMATOGRAPHY / Biotechnology Applications
for over 50 years. However, not all amino acids react equally well with ninhydrin, and the detection limit is B50 pmol (B6 ng) of amino acid derivative. The product of the reaction of ortho-phthaldialdehyde (OPA) with amino acids in the presence of thiol will fluoresce with excitation at 340 nm and emission at 450 nm. The reaction is fast (1–3 minutes), however, the OPA-derivatives are not stable. Secondary amino acids do not react and must first be converted into primary amino acids (oxidation with hypochlorite or chloramines-T (N-chloro-p-toluenesulfonamide). The detection limit in the case of postcolumn derivatization with OPA is B10 pmol (1.2 ng). Fluorescein has also been proposed for postcolumn derivatization of free amino acids but is not widely used due to the many problems associated with its application.
Conversion of amino acids to produce a diastereoisomeric peptide by reaction with an N-carboxyanhydride, e.g., L-Phe-N-carboxy-anhydride, has been used for determination of optical purity using RPC. Alternatively, for analysis of amino acid enantiomers without derivatization, two options are available: (1) chiral mobile phases such as the N,N-di-n-propyl-L-alanine–copper(II) complex can be used with reversed-phase columns, and (2) stationary phases with a covalently bound ligand capable of stereo recognition can be used. Such ligands include cyclodextrins, albumins, glycoproteins, and copper(II) complexes.
Dyes for precolumn derivatization The Edman reagent (phenylisothiocyanate) can be used to form thiohydantoin derivatives of primary and secondary amino acids, which can be separated using RPC (detection UV 254 nm). The detection limit is B1 pmol (B0.1 ng). OPA is also used for the precolumn derivatization. Detection limits (fluorescence) down to the femtomole level are possible. Some other dyes are of limited importance in precolumn derivatization. 4-(Dimethylamino)azobenzene-40 -sulfonyl chloride reacts with amino acids to produce a derivative that is stable at room temperature and can be detected at the 3–5 pmol level using detection at 436 nm. 4-(Dimethylamino)azobenzene-40 -isothiocyanate in combination with RPC has a detection limit of B5 pmol with detection at 436 nm. Fluorenylmethoxycarbonyl chloride forms derivatives of all amino acids, including proline. UV detection is possible at 260 nm, and fluorescence detection (excitation 266 nm, emission 305 nm) is also possible, the detection limit being B50 fmol in that case. 5-(Dimethylamino)naphthalene-1-sulfonyl chloride also reacts with both primary and secondary amines. The derivatives can be detected using UV at 254 nm or using fluorescence (excitation 385 nm, emission 460 nm). The sensitivity is in the low picomole range. Due to some problems with the specificity and the completeness of the reaction, the dye is rarely used.
The analysis of peptides and proteins naturally has some analogies with that of their main building blocks, amino acids (Figure 3). The analysis of proteomes, i.e., the entire gene product spectrum expressed by a given organism (cell) at a given time, is a very recent development in protein analysis. Proteome analysis is largely dominated by 2D gel electrophoresis (orthogonal separation of the protein mixture according to isoelectric point and mass) as a literature survey will show that still well over 90% of the pertinent separations are carried out using this method. However, the application of orthogonal LC–LC and LC–MS techniques for this purpose is gaining ground. For analysis of proteins and (synthetic) peptides, on the other hand, LC techniques are the method of choice (Figure 4). The problems encountered in analysis of free amino acids, namely the lack of an intrinsic detection method and the wide polarity range, are much less significant in the case of proteins/peptides. Since most of these molecules contain at least one aromatic amino acid and several peptide bonds, their UV detection at 280 nm and 214 nm, respectively, is straightforward. Since most peptides/ proteins are made for a mixture of polar and nonpolar amino acid residues, their separation using RPHPLC in a gradient of increasing organic solvent is the most commonly used approach. The resolution of RP-HPLC is high enough to show clear differences between a protein and its variants (reduced, deamidated form, etc.) and also between the correct product of a peptide synthesis and peptides where one or several addition steps have failed. A second important method for protein characterization and identification is the peptide map. For this purpose the protein is digested by a protease such as trypsin (tryptic map) or chymotrypsin, and the resulting peptide mix is analyzed using RP-HPLC. Even single
Enantiomeric Separations
The need to resolve the D- and L-enantiomers of amino acids has grown in recent years as it has been recognized that they differ in biological and physicochemical properties. Amino acid enantiomers cannot be resolved in achiral systems; it is therefore necessary to have a second chiral center either in the chromatographic system or to create one by derivatization in the molecule to be separated (Figure 2).
Peptides, Proteins, ‘Proteomics’
LIQUID CHROMATOGRAPHY / Biotechnology Applications 263
1
2
4
3
7 8
13, 14
6 5
9
10 11
12
15 16
0
20
40
60
80
Retent. time (min) Figure 2 Analysis of eight amino acid racemates. Chromatography conditions: column MCI GEL CRS10W, eluent 0.5 mmol l 1 copper (II) sulfate, flow rate 1.0 ml min 1, detection 254 nm, peaks 1 D-Ala, 2 L-Ala, 3 D-Pro, 4 D-Val, 5 L-Pro, 6 L-Val, 7 D-Leu, 8 D-Nle, 9 D-Tyr, 10 L-Leu, 11 D-Eth, 12 L-Tyr, 13 L-Nle, 14 D-Phe, 15 L-Eth, 16 L-Phe. (Reproduced with permission from Weston A and Brown Ph (1997) HPLC and CE – Principles and Practice. Academic Press, p. 61; & Elsevier.)
amino acid deviations can normally be detected as differences in the peak pattern given by the protein product and the variants. Peptide mapping is in addition very important for an indirect evaluation of the glycosylation pattern as the glycoform of a given peptide will show significant differences in the retention behavior compared with the nonglycosylated form. Finally, in combination with MS, the identity of the separated peptides can be determined and compared with the theoretical one in the case of proteins with known primary structure.
Carbohydrates Carbohydrate analysis has been slow to develop in comparison with that of other biologically important molecules. However, the more recent developments in biotechnology and renewable resources of raw materials have promoted interest in this diverse class
of compounds. Indeed, the term ‘carbohydrate’ encompasses a wide range of compounds from the simple building blocks (monosaccharides) to carbohydrate polymers, oligosaccharides, and polysaccharides and to various carbohydrate-containing species including glycoproteins, proteoglycans, nucleic acids, glycolipids, and antibiotics. Monosaccharides
There is a great variety in structure as well as chemical and physical properties of monosaccharides. There is no single method that is applicable to the qualitative and quantitative analysis of all monosaccharides; instead, the method must be chosen according to the chemical and physical characteristics of the solutes of interest. Borate complexes of monosaccharides can be separated using strong (quaternary amine, Q-type) anion-exchange columns. Alternatively, if the monosaccharide is acidic
264 LIQUID CHROMATOGRAPHY / Biotechnology Applications
or negatively charged at high pH, anion exchangers can be used directly. Cation-exchange chromatography is normally used for analysis of amino sugars and – using calcium as counterion – alditols and monosaccharides. Oligosaccharides
Oligosaccharides are traditionally defined as polymers of monosaccharides containing from two to ten
A
Absorbance at 215 nm
0.10
B
C
0.05
0.0 0
3
0
4 0 Time (min)
6
Figure 3 Determination of protein purity. Chromatographic conditions: column Hy-Tach micropellicular C-18 silica, eluent A 0.1% TFA in water, eluent B 95% acetonitrile in water containing 0.1% TFA, flow rate 2.0 ml min 1, sample A 40 mg carbonic anhydrase (gradient 15 to 55% B in 3 min), B 40 mg L-asparaginase (gradient 30 to 40% B in 4 Lmin), C 40 mg myoglobin (gradient 23 to 45% B in 6 min). (Reproduced with permission from Horvath C and Nikelly JG (eds.) (1990) Analytical Biotechnology, ACS Symposium Series 434; & American Chemical Society.)
residues, although this range is often extended to 25 as naturally occurring polysaccharides rarely contain less than 25 repeat units. Anion-exchange resins with acetate, hydroxyl, or chloride counterions are usually used for oligosaccharide analysis. For a homologous series, the elution increases with increasing chain length. In an attempt to overcome the problems of interconversion of the terminal reducing residues often encountered in anion-exchange chromatography of oligosaccharides, cation-exchange resins, in particular sulfonated poly(styrene–divinyl–benzene) materials, have been used for oligosaccharide separations, with water as the mobile phase. The effect of the counterion, e.g., lithium, barium, potassium, silver, or lead, is considerable as the separation mechanism is not purely cation exchange but also encompasses ligand exchange. Gel filtration chromatography is another very useful technique for analysis of oligosaccharides. By a judicious choice of the stationary phase, a resolution of up to DP 15 can be achieved. The use of RPC for oligosaccharide analysis, on the other hand, has in general been limited to smaller oligomers owing to problems of solubility in the typical hydro-organic mobile phases. Normal-phase chromatography using stationary phases with amino or cyano functionalities has been used extensively for oligosaccharide analysis, again with aqueous acetonitrile mobile phases. However, unlike in RPC, elution takes place in a gradient of increasing water content of the mobile phase. Higher oligomers or polysaccharide material may be bound irreversibly to the column or have excessively long elution times.
r t-PA reference standard arginine at position 275
Mutant form of r t-PA with Glutamic acid at position 275 0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
Time (min) Figure 4 Comparison of the tryptic map of a recombinant tissue plasminogen activator reference standard and a mutant form with a glutamic acid residue instead of the normal arginine residue at position 275. (Reproduced with permission from Weston A and Brown Ph (1997) HPLC and CE – Principles and Practice, Academic Press, p. 37; & Elsevier.)
LIQUID CHROMATOGRAPHY / Biotechnology Applications 265 High molecular mass
I
6
II
III IV
7 1 3 12 8 13 11 15 10 9 14
5
4
2 2 3
1
5
4 A 3
6 Time (h)
9
Figure 5 Gel filtration chromatography of starch-derived oligosaccharides on a Bio-Gel P2 column. Peak numbers refer to the degree of polymerization.
B
C
Polysaccharides
The number of liquid chromatographic methods available for analysis of polysaccharides is limited compared with those for monomers or oligomers. Polysaccharides comprise a diverse group of complex macromolecules that may range in molecular mass from several thousand to several million Daltons and may be composed of neutral, acidic, or basic monomers or mixtures thereof arranged in a variety of structures. As polysaccharides are polydisperse macromolecules, gel filtration is often the first liquid chromatographic method to be used for their characterization. As the separation mechanism is one based on solute size, columns can be calibrated with polysaccharides of known molecular masses, and estimates for unknowns can be made. IEC is used to assess the homogeneity of a polysaccharide, often in combination with gel filtration chromatography, to separate molecules of similar size on the basis of differences in their charges (Figure 5). Carbohydrate-Containing Macromolecules, Glycoproteins
The methods previously discussed for mono-, oligo-, and polysaccharides are also applicable to analysis of carbohydrate-containing macromolecules. For example, proteoglycans, which consist of a central core protein to which a number of glycosaminoglycan chains are attached, which carry a significant negative charge due to a high concentration of carboxyl and sulfate ester groups, can be separated using anion-exchange chromatography (Figure 6). Of increasing importance in medical biotechnology is analysis of the glycosylation pattern, i.e., the composition and structure of the oligosaccharide chains of glycoproteins. While the amino acid sequence is encoded by the genetic code and can hence be transferred by genetic engineering from one
D
E
12
14
16
18 20 time (min)
22
24
Figure 6 HPAEC–PAD mapping of N- and O-glycans of glycoproteins after release by hydrazinolysis. (Reproduced with permission from Animal cell technology: products from cells, cells as products in Proceedings of the 16th ESACT Meeting, April 25–29, Lugano, Switzerland ; & Kluwer Academic Publishers.)
species to the next, glycosylation is a post-translatorial event and hence shows pronounced differences from species to species and even between the different cell types of a given organism. At the same time glycosylation does have significant consequences for the biological activity as well as for the pharmacokinetics and -dynamics. A typical procedure in analysis of glycosylation patterns is comparison of the peptide map of the native glycoprotein and of that of the residual polypeptide structure after enzymatic release of the sugar residues together with a direct analysis of the released glycans using high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). Detection Methods
In general carbohydrates do not have a UV chromophore and do not fluoresce. Monosaccharides do absorb in the far UV, with the absorbance maxima at B188 nm. Detection is normally performed at wavelengths between 192 and 200 nm, where there is less residual noise in the detector signal, if the absorbance of the solvent permits this. The UV
266 LIQUID CHROMATOGRAPHY / Biotechnology Applications 1 2 8
5
10
7 3 4
13 6 9
11
12
automated versions of the spectrophotometric assays traditionally used for identification and quantification of carbohydrates. Methods of detection for total carbohydrate include the following: L-cysteine–sulphuric acid- and phenol–sulfuric acid-based assays for neutral carbohydrates, the carbazole assay or Warren assay for acid carbohydrates, the Elson Morgan assay for basic carbohydrates, and the 3,5-dinitrosalicylic acid assay for reducing groups.
Organic Acids
24 Time (min)
48
Figure 7 Ion exclusion isocratic separation of organic acids. Peaks: 1 chloride, 2 oxalate, 3 pyruvate, 4 tartrate, 5 malonate, 6 lactate, 7 malate, 8 acetate, 9 isocitrate, 10 citrate, 11 b-hydroxyn-butyrate, 12 succinate, 13 proprionate. (Reproduced with permission from Dionex Product Selection Guide 1991.)
response is dependent upon the carbonyl group and will differ for the individual monosaccharides, fructose and glucose being the two extremes, with the response of fructose being six times that of glucose. Differential refractive index detection has often been used in the past; however, the method is not particularly sensitive and is only suitable for isocratic separations. Increasingly, PAD is being used as the universal method in the LC of mono- and oligosaccharides (Figure 7). The carbohydrate is oxidized at a gold electrode at a pH greater than 11, with the electrode surface being reduced back to gold using a negative potential sequence. In addition, carbohydrates can be derivatized to produce UV or fluorescent species to improve detection. As with the amino acids, both precolumn and postcolumn derivatization are used. Precolumn derivatization influences the chromatographic behavior of the compounds. For UV detection, phenylisocyanate derivatization can be used together with RP-HPLC or benzoyl chloride and 4-bromobenzoyl chloride derivatization together with normal-phase chromatography. When fluorescence detection is preferred, dansyl hydrazine and aminopyridine derivatives can be analyzed using RPC. Postcolumn derivatization is normally preferred for carbohydrate analysis as it is applicable to mono-, oligo-, and polysaccharides. It also improves the detection limit, and of course analysis selectivity, but without altering the intrinsic separation characteristics of the carbohydrates by introducing a group or groups that may dominate the separation process. The most common derivatization reactions are
Short-chain mono-, di-, and polycarboxylic acids are of significance to scientists studying amino acid degradation, metabolites of carbohydrate oxidation, and lipid breakdown. They can be indicators of human metabolic disorders and of importance in the biotechnology of food. LC is now the preferred method of analysis for these analytes irrespective of the sample origin – biological fluid, fermentation broth, or food. IEC, or perhaps more correctly ion-exclusion chromatography using sulfonated polymeric resins, has become the method of choice for separating organic acids. The stationary phase can withstand extremes of pH, and the separation of the organic acids is normally according to pKa values, but partition effects can also occur. Dilute acids are used as the mobile phase. Normally only isocratic mobile phases are required, with the addition of a small quantity of an organic modifier (usually acetonitrile) for analysis of nonpolar acids. In the case of weak organic acids, ion pairing agents can be used and the analysis can then also be achieved using RP-HPLC. However, this separation system is limited to certain organic acids, and the reproducibility can be poor. Detection can be achieved at nanogram or picogram levels, using electrochemical and conductivity detectors.
Monitoring Fermentations and Fermentation Products LC continues to be an important tool in fermentation monitoring. Using modern technology, the methods used are sufficiently fast and robust even for fermentation control, for example, to supervise nutrient addition and hence to maintain an optimum environment for the production organisms. Carbohydrates, amino acids, and the content of organic acids, all critical to either the production or the inhibition of production, can all be monitored as described above. In the production of wine, monitoring the pyruvic acid content during malo-lactic fermentation gives an
LIQUID CHROMATOGRAPHY / Clinical Applications 267
indication of the health of the bacteria, a pyruvate being an indirect measure of NADH, the reducing co-factor that stimulates bacterial growth. Malates and tartrates are also simultaneously monitored, being indicators of the maturity of the fermentation process. Lactose fermentation can yield lactic, acetic, pyruvic, formic, propionic, and butyric acids, which are indicators of the flavor and stability of dairy products. All these acids can be determined in one LC-analysis run. In the food industry LC is frequently used to determine the quality of the finished product. For example, it has been shown that the flavor quality of grape juice is directly linked to the amount and spectrum of organic acids present. Likewise, in the production of sour cream by fermentation, the quality of the finished product quality is related directly to the organic acid profile. Last, but not least and as pointed out above, LC techniques are also crucial in determining the quality of recombinant protein products. See also: Carbohydrates: Overview; Sugars – Chromatographic Methods. Chromatography: Overview; Principles. Derivatization of Analytes. Electrophoresis: Two-Dimensional Gels; Proteins. Fluorescence: Derivatization; Fluorescence Labeling. Ion Exchange: Overview; Principles; Isolation of Biopolymers. Liquid Chromatography: Overview; Principles; Column Technology; Mobile Phase Selection; Normal Phase; Reversed Phase; Size Exclusion; Chiral; Affinity Chromatography; Multidimensional; Instrumentation; Liquid Chromatography–Mass Spectrometry; Amino Acids; Clinical Applications. Micro Total Analytical Systems. Peptides. Pharmaceutical Analysis: Overview. Process Analysis: Chromatography; Bioprocess Analysis.
Proteins: Traditional Methods of Sequence Determination. Proteomics.
Further Reading Chaplin MF (1986) Monosaccharides. In: Chaplin MF and Kennedy JF (eds.) Carbohydrate Analysis – A Practical Approach, pp. 37–69. Oxford IRL Press. El Rassi Z (ed.) (1995) Carbohydrate Analysis. Journal of Chromatography Library, vol. 58. Elsevier. Heftmann E (ed.) (1991) Chromatography, 5th edn., Journal of Chromatography Library, vol. 51. Elsevier. Horvath C and Nikelly JG (eds.) (1990) Analytical Biotechnology. ACS Symposium Series 434. Washington, DC: American Chemical Society. Kastner M (ed.) (2000) Protein Liquid Chromatography. Journal of Chromatography Library, vol. 67. Elsevier. Keller R, Lottspeich F, Meaer HE (eds.) (1999) Microcharacterization of Proteins. Wiley–VCH. Lloyd LL, Warner FP, and Kennedy JF (1990) The application of HPLC in biotechnology. In: Biotechnology International, pp. 221–226. London: Century Press. Schwarzenbach R (1982) High-performance liquid chromatography of carboxylic acids. Journal of Chromatography 251: 339–358. Sofer G and Hagel L (1997) Handbook of Process Chromatography – A Guide to Optimization, Scale-up and Validation. Academic Press. Tukova J (ed.) (1993) Bioaffinity Chromatography, 2nd completely revised edn., Journal of Chromatography Library, vol. 55. Elsevier. Weston A and Brown Ph (1997) HPLC and CE – Principles and Practice. Academic Press. White CA and Kennedy JF (1986) Oligosaccharides. In: Chaplin MF and Kennedy JF (eds.) Carbohydrate Analysis – A Practical Approach, pp. 37–69. Oxford IRL Press.
Clinical Applications R A Sherwood, King’s College Hospital, London, UK & 2005, Elsevier Ltd. All Rights Reserved. This article is a revision of the previous-edition article by R A Sherwood and B F Rocks, pp. 2677–2685, & 1995, Elsevier Ltd.
Introduction The diagnosis and monitoring of many disease states relies on the detection and quantitation of a particular compound(s) or metabolite(s) in body fluids, usually blood or urine. A wide variety of techniques are used in the clinical chemistry laboratory with spectrophotometric and immunoassay methods on automated analyzers measuring single compounds
predominating. In some instances a family of compounds may need to be measured and it is in these situations where the chromatographic techniques are important to avoid the necessity for a separate assay for each component of interest in the mixture. Initially chromatography was carried out using paper or slab gels. The use of columns to contain the stationary phase came more recently with the advent of high-performance liquid chromatography (HPLC). Typical applications of HPLC in clinical chemistry include: 1. Biogenic amines (a) Catecholamines and metabolites (b) Serotonin and metabolites