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A reappreciation of alumina in high-performance liquid chromatography
100
tility. The less expensive, dedicated LC instrument is more likely to include pump, injector, column, detector and data processor all in one unit. ‘Personal’ instruments It is not presumptuous at this time to predict the evolution of the ‘personal liquid chromatograph’ and the ‘personal gas chromatograph’. It is likely that the chemist of the future will have his or her own personal GC, LC, and even UV and IR instrument on the bench for his or her exclusive use, Such thoughts, of course, may at this time be considered extravagant, probably as extravagant as those computer technologists who fifteen years ago predicted the possibility of the personal computer. Progress is inexorable! The personal analytical instruments will, in due course, be with us and ultimately be found as companions to the personal computer that is already in the classrooms of our high schools. The price must be
trendsin analyticalchemistry, vol. 4, no. (I,1985
right, the performance must be good, and they must be reliable. It is only a matter of time! Many workers in the field of chromatography and perhaps even some instrument companies feel that the techniques of gas and liquid chromatography have reached a plateau and will not change significantly in the future. It is more likely that it is the imagination of the engineers and scientists themselves and not the technique that has reached this limitation. Nothing is ever static, progress will continue providing better, faster, more accurate and less expensive instruments in the future.
R. P. W. Scott is at the Perkin-Elmer Norwalk, CT06856, U.S.A.
Corporation, Main Avenue,
A reappreciation of alumina in highperformance liquid chromatography Hugo Billiet, Claude Laurent and Leo de Galan Delft,The Netherlands
Introduction The use of alumina as the stationary phase material in modern liquid chromatography has steadily decreased during the last ten years. In classical adsorption chromatography (liquid-solid chromatography) it must compete with silica and both materials have been largely superseded by chemically modified, depolarized silicas. Like silica, alumina must be considered as a typical polar adsorbent and most separations proceed in the same way on the two oxides. However, whereas silica is only active through its surface hydroxyl groups, alumina possesses two alternative possibilities for interaction with solutes. The acidic sites of alumina adsorb basic solutes through nucleophilic interaction. The same sites may also form charge transfer complexes with typical electron donors such as aromatic solutes’. On the other hand, acidic solutes may interact with basic sites on the alumina surface through transfer of a proton leading to chemi0165-9936/85/$02.00.
sorption. The important example is water, which through chemisorption gives rise to two hydroxyl groups on the alumina surface. In general then, alumina interacts strongly with polarized molecules; so much in fact, that its applicability is often limited by the adverse influence of chemisorption on the peak shape. The crystalline alumina generally used in chromatography is known as y-alumina. The Al-O bond has a stronger ionic character than the Si-0 bond. From the nature of the Al-O bond and the electrostatic surface behaviour it might be suspected that in silanized alumina the surface Al-0-Si bond is less stable than the Si-0-Si bond. Indeed, our experience has been that it was impossible to modify the alumina surface into a reversed-phase material similar to modified silicas. Ion-exchange properties of alumina Alumina acts as a typical ion-exchanger, but it is also amphoteric in nature so that its ion-exchange properties will be strongly pH-dependent. This amphoteric character can be explained by the presence of two distinct types of hydroxyl groups on the alumina surface (see Fig. 1 for a simplified picture). On 0
Elsevier Science Publishers B.V.
101
trendsin analyticalchemistry, vol. 4, no. 4,1985
0
CL-
\
-
acidic alumina
0
/
\ H-
AL-
AL-
/ -0
\ ‘0 -41
/ \
0 --
NaOH
HCL
0 OH-
H 0
/
\ --AL/
d
basic alumina
\ 0 Na-
-0
AL-
/ \
Fig. 1. Surface behaviour of alumina in basic and acidic media. (From ref. I, with permission.)
the one hand, there will be hydroxyl groups chemisorbed onto acidic sites (Al atoms). On the other hand, there will be protons chemisorbed onto oxygen atoms’. When neutral alumina is washed with a sodium hydroxide solution, the chemisorbed protons will be neutralized and replaced by more loosely bound sodium ions. The sodium ions can exchange with other cations and are responsible for the cation-exchange properties of alkaline alumina at higher pH. Conversely, when the alkaline alumina is washed with hydrochloric acid, the protons of the acid produce two effects. They desorb the hydroxyl groups, which are then replaced by chloride ions to give rise in the anion-exchange properties of acidic alumina. The protons also replace the sodium cations attached to oxygen atoms. As a result of these washings, alumina has cationand anion-exchange properties over fairly broad and overlapping pH-ranges. Nevertheless, it is possible to define a pH where the net charge of the surface is zero (zero point of charge, ZPC). At lower pH the net charge is positive, at higher pH it is negative. Generally, the ZPC of an aqueous suspension
of the oxide does not differ much from that of the solid metal hydroxide. However, the structure of the oxide and the presence of other ions co-precipitated during its preparation as well as added through buffers exert a large influence on the ZPC. Pure y-alumina has a ZPC-value of 9.2, but in the presence of H,PO, ions the ZPC shifts to 6.2. Other examples are: a ZPC of 9.2 in carbonate buffer, 6.5 in acetate and 3.5 in citrate buffer. This means that the anion of the weak acid is responsible for the acidic shift. The separations of basic solutes on alumina It is known from the literature that chromatography of very basic solutes on silica or reversed-phase materials often results in peaks with tails or poor peak shapes, even when an ion-pairing reagent is added. The high pH of the eluent and the addition of amine-like silanol blocking agents attacks the silica structure and ruins the column efficiency. By contrast, alumina has proven to be very stable at extreme pH. Experiments have shown that the aluminum content (measured by inductively coupled plasma) of 0.1 M sodium hydroxide percolated through an analytical column did not exceed 10 ppm. This means less than 1% dissolution of column material after one month of continuous use of pH = 13. In classical ion-exchange chromatography, retention can be controlled in several ways. The nature of the buffer ion and the ionic strength of the eluent have a strong influence on the overall retention, but no specific influence on the relative retention of two compounds. To a certain extent, selectivity can be manipulated by a judicious choice of the pH of the mobile phase. However, apart from the practical problem of adjusting and maintaining a precise pH, the applicability is rather limited. Another, much more flexible way to influence retention and selectivity in ion-exchange chromatography is to add appreciable amounts of organic modifier to the aqueous solvent2. In interpreting the results, we can ignore the effect of swelling that occurs with organic ion exchange resins. The variation in solute retention observed by adding methanol, acetonitrile or tetrahydrofuran can be interpreted by a change in ionization and solvation of the solutes and the buffer cation (see Fig. 2). The appearance of a maximum suggests that there are at least two competitive mechanisms. Initially, at low modifier content, there is a general and gradual increase in retention. If we suppose that the solute cations compete with the buffer cations for the active sites on the alumina surface, in the presence of organic modifier the tetra-alkyl ammonium ions become less active. The enhanced solvation of the buffer cation (preferentially from organic nature) weak-
102
trends in analytical chemistry, vol. 4, no. 4~1985
I
0
1
10
I
20
1
Elifier
1
40
content
(%) -
Fig. 2. Influence of organic modifiers on retention behaviour of n-alkylamines (indicated by their carbon number). Methanol (-), acetonitrile (--) and tetrahydrofuran (. . .).
ens its competition with the solute ions and accounts for the increasing retention. The decrease in retention can be attributed to reduced solute ionization in water-organic mixtures with a high organic content. This phenomenon seems to depend on the nature of the solute and buffer cation and on the pH selected for the buffer in combination with the pK, value of the solute. The separation of heroin and opium compounds is a typical example of how selectivity can be adjusted in such a system (see Figs. 3 and 4)3. Some rules of thumb can be derived from the heroin example. If the pK, value of the solutes is between 6 and 9, than the pH of the buffer should be around 5 which means that citrate is perfect (ZPC is at pH 3.5). The retention can be manipulated by the chain length of the tetra-alkyl ammonium and the type and concentration of organic modifier. The selectivity effect is unpredictable and should be measured empirically. Temperature can also play an important role as its influence on solute ionization can be important. The separation of proteins on alumina The separation of proteins is one of the most challenging areas in modern liquid chromatography and currently receives large attention4. Various new materials have been developed during the last ten years, mostly directed to ion-exchange and size exclusion mechanisms. In this context, alumina presents an attractive alternative as both mechanisms can be combined. For the separation of proteins, the concept of ZPC has a close parallel in the definition of the isoelectric point (pl) where the net charge of the polyelectrolytic protein is zero. For pH > pl the net charge of the protein is negative, for pH < pl it is positive. Consequently, the relative values of the ZPC of alumina
I
24
1
20
I
I
I
I
t
16
12
8
4
0
Retention
time
(min)
Fig. 3. Standard chromatogram of heroin and opium compounds. Conditions: citric acid and tetrabutylammonium hydroxide (0.001 M) pH 5.5, in methanol-water (3565). Column (20 cm x 4.6 mm I.D.) packed with Spherisorb A5Y. Temperature, 35 “C. Peaks: 1 = papaverine; 2 = narcotine; 3 = heroin; 4 = procaine; 5 = acetylcodeine; 6 = 6monoacetylmorphine; 7 = codeine; 8 = morphine; 9 = strychnine.
and the pZ of a protein are of paramount importance for the ion-exchange retention observed. Two situations can be distinguished. If ZPC < pH < pl, then the positively charged protein is in contact with negatively charged alumina and will be retained by cation exchange. If, on the other hand, pZ < pH < ZPC the negatively charged protein experiences the presence of a positive alumina surface and will be retained by anion exchange. In either case, no ion-exchange retention should be observed for pH values outside this range. We may also expect the largest variation in retention, when the pH is varied around either the ZPC of alumina or the pZ of the protein. So far, the argument has focussed on the ion-exchange properties of alumina. Obviously, this is not the only retention mechanism possible for proteins.
tren& in analytical chemistry,
103
vol. 4, no. 4,198s
6
L
I
b
a
2
::A 7
1 2 3
3
0
9
5
A
”
:
I
I
0
5
I
I
15
10 Retention
time (min)
0
z&k% Retention
10 time
(min)
8
6 Retention
4
2)
0
timecmin)
Fig. 4. Influence of organic modifier on the separation of alkaloids on alumina in the ion-exchange mode. Conditions: citric acid and tetramethyl ammonium hydroxide (0.01 M) pH 6, in (a) water and (b) acetonitrile (30:70). Column (20 cm x 4.6 mm I.D.) packed with Spherisorb AIOY. W detection at 254 nm. Peaks: 1 = dimethylmorphine; 2 = codeine; 3 = morphine.
The pore size of commercial alumina (13 nm for Spherisorb) leads to size-exclusion effects for proteins with molecular mass between 1000 and 20,000, provided that the electrostatic attraction of the charged proteins is overcome by the addition of salts to the eluent. A typical example of a protein separation on alumina is given in Fig. 5, where various proteins are separated at pH = 9, obtained with a phosphate buffer that renders the ZPC equal to 6.5. Phosphate buffers can be used with advantage, because its successive stages of ionization allow the coverage of a pH-range from 6.5 (the ZPC of alumina) up to 11. Conclusions l Alumina is an amphoteric ion exchanger which acts as a cation exchanger in a basic eluent and as anion exchanger at acidic pH. The transition takes place in a pH value where the net charge of the surface is zero (ZPC). The ZPC moves along the pH scale depending on the nature of the buffer used. l The ion exchange selectivity can be influenced by adding organic modifiers such as methanol, acetonitrile or tetrahydrofuran to the eluent. l Alumina is an interesting stationary phase for protein separations, especially in the case of basic proteins. The combination of the size-exclusion and
Fig. 5. Chromatogram of some standard proteins. Mobile phase: 0.25 M Na2HP0, (pH 9). Solutes: 1 and 2 = bovine albumin; 3 = ovalbumin; 4 = myoglobin; 5 = unknown; 6 = arginine vasopressin; 7 = trypsinogen; 8 = lysozyme; 9 = chymotrypsinogen.
the ion-exchange mechanisms allows excellent separations to be obtained by simple means. l Basic solutes can easily be chromatographed on alumina resulting in symmetrical peaks. Columns of high efficiency can be packed in methanol and usually have a high permeability.
References This article is an abstract of Claude Laurent’s Ph.D. thesis entitled: A reappreciation of alumina in high pressure liquid chromatography. The work was done at the laboratory of Analytical Chemistry of the Delft University of Technology under the supervision of Prof Dr L. de Galan. The thesis contains the following papers: 1 C. J. C. M. Laurent, H. A. H. Billiet and L. de Galan, Chromatographia, 17 (1983) 253. (On the use of alumina in HPLC with aqueous mobile phases at extreme PH.). 2 C. J. C. M. Laurent, H. A. H. Billiet and L. de Galan, Chromatographia, 17 (1983) 394. (The use of organic modifiers in ion-exchange chromatography on alumina. The separation of basic drugs.). 3 C. J. C. M. Laurent, H. A. H. Billiet and L. de Galan, J. Chromatogr., 285 (1984) 161-170. (High-performance liquid chromatography of heroin samples on alumina by ion exchange in mixed aqueous-organic mobile phases.). 4 C. J. C. M. Laurent, H. A. H. Billiet, L. de Galan, F. A. Buytenhuys and F. P. B. van der Maeden, J. Chromatogr., 287 (1984) 45-54. (High-performance liquid chromatography of proteins on alumina.). H. Billiet, C. Laurent and L. de Galan are at the Laboratorium voor Analytische Scheikunde, Technische Hogeschool Delft, Jaffalaan 9,2628 BX Delft, The Netherlands.
tility. The less expensive, dedicated LC instrument is more likely to include pump, injector, column, detector and data processor all in one unit. ‘Personal’ instruments It is not presumptuous at this time to predict the evolution of the ‘personal liquid chromatograph’ and the ‘personal gas chromatograph’. It is likely that the chemist of the future will have his or her own personal GC, LC, and even UV and IR instrument on the bench for his or her exclusive use, Such thoughts, of course, may at this time be considered extravagant, probably as extravagant as those computer technologists who fifteen years ago predicted the possibility of the personal computer. Progress is inexorable! The personal analytical instruments will, in due course, be with us and ultimately be found as companions to the personal computer that is already in the classrooms of our high schools. The price must be
trendsin analyticalchemistry, vol. 4, no. (I,1985
right, the performance must be good, and they must be reliable. It is only a matter of time! Many workers in the field of chromatography and perhaps even some instrument companies feel that the techniques of gas and liquid chromatography have reached a plateau and will not change significantly in the future. It is more likely that it is the imagination of the engineers and scientists themselves and not the technique that has reached this limitation. Nothing is ever static, progress will continue providing better, faster, more accurate and less expensive instruments in the future.
R. P. W. Scott is at the Perkin-Elmer Norwalk, CT06856, U.S.A.
Corporation, Main Avenue,
A reappreciation of alumina in highperformance liquid chromatography Hugo Billiet, Claude Laurent and Leo de Galan Delft,The Netherlands
Introduction The use of alumina as the stationary phase material in modern liquid chromatography has steadily decreased during the last ten years. In classical adsorption chromatography (liquid-solid chromatography) it must compete with silica and both materials have been largely superseded by chemically modified, depolarized silicas. Like silica, alumina must be considered as a typical polar adsorbent and most separations proceed in the same way on the two oxides. However, whereas silica is only active through its surface hydroxyl groups, alumina possesses two alternative possibilities for interaction with solutes. The acidic sites of alumina adsorb basic solutes through nucleophilic interaction. The same sites may also form charge transfer complexes with typical electron donors such as aromatic solutes’. On the other hand, acidic solutes may interact with basic sites on the alumina surface through transfer of a proton leading to chemi0165-9936/85/$02.00.
sorption. The important example is water, which through chemisorption gives rise to two hydroxyl groups on the alumina surface. In general then, alumina interacts strongly with polarized molecules; so much in fact, that its applicability is often limited by the adverse influence of chemisorption on the peak shape. The crystalline alumina generally used in chromatography is known as y-alumina. The Al-O bond has a stronger ionic character than the Si-0 bond. From the nature of the Al-O bond and the electrostatic surface behaviour it might be suspected that in silanized alumina the surface Al-0-Si bond is less stable than the Si-0-Si bond. Indeed, our experience has been that it was impossible to modify the alumina surface into a reversed-phase material similar to modified silicas. Ion-exchange properties of alumina Alumina acts as a typical ion-exchanger, but it is also amphoteric in nature so that its ion-exchange properties will be strongly pH-dependent. This amphoteric character can be explained by the presence of two distinct types of hydroxyl groups on the alumina surface (see Fig. 1 for a simplified picture). On 0
Elsevier Science Publishers B.V.
101
trendsin analyticalchemistry, vol. 4, no. 4,1985
0
CL-
\
-
acidic alumina
0
/
\ H-
AL-
AL-
/ -0
\ ‘0 -41
/ \
0 --
NaOH
HCL
0 OH-
H 0
/
\ --AL/
d
basic alumina
\ 0 Na-
-0
AL-
/ \
Fig. 1. Surface behaviour of alumina in basic and acidic media. (From ref. I, with permission.)
the one hand, there will be hydroxyl groups chemisorbed onto acidic sites (Al atoms). On the other hand, there will be protons chemisorbed onto oxygen atoms’. When neutral alumina is washed with a sodium hydroxide solution, the chemisorbed protons will be neutralized and replaced by more loosely bound sodium ions. The sodium ions can exchange with other cations and are responsible for the cation-exchange properties of alkaline alumina at higher pH. Conversely, when the alkaline alumina is washed with hydrochloric acid, the protons of the acid produce two effects. They desorb the hydroxyl groups, which are then replaced by chloride ions to give rise in the anion-exchange properties of acidic alumina. The protons also replace the sodium cations attached to oxygen atoms. As a result of these washings, alumina has cationand anion-exchange properties over fairly broad and overlapping pH-ranges. Nevertheless, it is possible to define a pH where the net charge of the surface is zero (zero point of charge, ZPC). At lower pH the net charge is positive, at higher pH it is negative. Generally, the ZPC of an aqueous suspension
of the oxide does not differ much from that of the solid metal hydroxide. However, the structure of the oxide and the presence of other ions co-precipitated during its preparation as well as added through buffers exert a large influence on the ZPC. Pure y-alumina has a ZPC-value of 9.2, but in the presence of H,PO, ions the ZPC shifts to 6.2. Other examples are: a ZPC of 9.2 in carbonate buffer, 6.5 in acetate and 3.5 in citrate buffer. This means that the anion of the weak acid is responsible for the acidic shift. The separations of basic solutes on alumina It is known from the literature that chromatography of very basic solutes on silica or reversed-phase materials often results in peaks with tails or poor peak shapes, even when an ion-pairing reagent is added. The high pH of the eluent and the addition of amine-like silanol blocking agents attacks the silica structure and ruins the column efficiency. By contrast, alumina has proven to be very stable at extreme pH. Experiments have shown that the aluminum content (measured by inductively coupled plasma) of 0.1 M sodium hydroxide percolated through an analytical column did not exceed 10 ppm. This means less than 1% dissolution of column material after one month of continuous use of pH = 13. In classical ion-exchange chromatography, retention can be controlled in several ways. The nature of the buffer ion and the ionic strength of the eluent have a strong influence on the overall retention, but no specific influence on the relative retention of two compounds. To a certain extent, selectivity can be manipulated by a judicious choice of the pH of the mobile phase. However, apart from the practical problem of adjusting and maintaining a precise pH, the applicability is rather limited. Another, much more flexible way to influence retention and selectivity in ion-exchange chromatography is to add appreciable amounts of organic modifier to the aqueous solvent2. In interpreting the results, we can ignore the effect of swelling that occurs with organic ion exchange resins. The variation in solute retention observed by adding methanol, acetonitrile or tetrahydrofuran can be interpreted by a change in ionization and solvation of the solutes and the buffer cation (see Fig. 2). The appearance of a maximum suggests that there are at least two competitive mechanisms. Initially, at low modifier content, there is a general and gradual increase in retention. If we suppose that the solute cations compete with the buffer cations for the active sites on the alumina surface, in the presence of organic modifier the tetra-alkyl ammonium ions become less active. The enhanced solvation of the buffer cation (preferentially from organic nature) weak-
102
trends in analytical chemistry, vol. 4, no. 4~1985
I
0
1
10
I
20
1
Elifier
1
40
content
(%) -
Fig. 2. Influence of organic modifiers on retention behaviour of n-alkylamines (indicated by their carbon number). Methanol (-), acetonitrile (--) and tetrahydrofuran (. . .).
ens its competition with the solute ions and accounts for the increasing retention. The decrease in retention can be attributed to reduced solute ionization in water-organic mixtures with a high organic content. This phenomenon seems to depend on the nature of the solute and buffer cation and on the pH selected for the buffer in combination with the pK, value of the solute. The separation of heroin and opium compounds is a typical example of how selectivity can be adjusted in such a system (see Figs. 3 and 4)3. Some rules of thumb can be derived from the heroin example. If the pK, value of the solutes is between 6 and 9, than the pH of the buffer should be around 5 which means that citrate is perfect (ZPC is at pH 3.5). The retention can be manipulated by the chain length of the tetra-alkyl ammonium and the type and concentration of organic modifier. The selectivity effect is unpredictable and should be measured empirically. Temperature can also play an important role as its influence on solute ionization can be important. The separation of proteins on alumina The separation of proteins is one of the most challenging areas in modern liquid chromatography and currently receives large attention4. Various new materials have been developed during the last ten years, mostly directed to ion-exchange and size exclusion mechanisms. In this context, alumina presents an attractive alternative as both mechanisms can be combined. For the separation of proteins, the concept of ZPC has a close parallel in the definition of the isoelectric point (pl) where the net charge of the polyelectrolytic protein is zero. For pH > pl the net charge of the protein is negative, for pH < pl it is positive. Consequently, the relative values of the ZPC of alumina
I
24
1
20
I
I
I
I
t
16
12
8
4
0
Retention
time
(min)
Fig. 3. Standard chromatogram of heroin and opium compounds. Conditions: citric acid and tetrabutylammonium hydroxide (0.001 M) pH 5.5, in methanol-water (3565). Column (20 cm x 4.6 mm I.D.) packed with Spherisorb A5Y. Temperature, 35 “C. Peaks: 1 = papaverine; 2 = narcotine; 3 = heroin; 4 = procaine; 5 = acetylcodeine; 6 = 6monoacetylmorphine; 7 = codeine; 8 = morphine; 9 = strychnine.
and the pZ of a protein are of paramount importance for the ion-exchange retention observed. Two situations can be distinguished. If ZPC < pH < pl, then the positively charged protein is in contact with negatively charged alumina and will be retained by cation exchange. If, on the other hand, pZ < pH < ZPC the negatively charged protein experiences the presence of a positive alumina surface and will be retained by anion exchange. In either case, no ion-exchange retention should be observed for pH values outside this range. We may also expect the largest variation in retention, when the pH is varied around either the ZPC of alumina or the pZ of the protein. So far, the argument has focussed on the ion-exchange properties of alumina. Obviously, this is not the only retention mechanism possible for proteins.
tren& in analytical chemistry,
103
vol. 4, no. 4,198s
6
L
I
b
a
2
::A 7
1 2 3
3
0
9
5
A
”
:
I
I
0
5
I
I
15
10 Retention
time (min)
0
z&k% Retention
10 time
(min)
8
6 Retention
4
2)
0
timecmin)
Fig. 4. Influence of organic modifier on the separation of alkaloids on alumina in the ion-exchange mode. Conditions: citric acid and tetramethyl ammonium hydroxide (0.01 M) pH 6, in (a) water and (b) acetonitrile (30:70). Column (20 cm x 4.6 mm I.D.) packed with Spherisorb AIOY. W detection at 254 nm. Peaks: 1 = dimethylmorphine; 2 = codeine; 3 = morphine.
The pore size of commercial alumina (13 nm for Spherisorb) leads to size-exclusion effects for proteins with molecular mass between 1000 and 20,000, provided that the electrostatic attraction of the charged proteins is overcome by the addition of salts to the eluent. A typical example of a protein separation on alumina is given in Fig. 5, where various proteins are separated at pH = 9, obtained with a phosphate buffer that renders the ZPC equal to 6.5. Phosphate buffers can be used with advantage, because its successive stages of ionization allow the coverage of a pH-range from 6.5 (the ZPC of alumina) up to 11. Conclusions l Alumina is an amphoteric ion exchanger which acts as a cation exchanger in a basic eluent and as anion exchanger at acidic pH. The transition takes place in a pH value where the net charge of the surface is zero (ZPC). The ZPC moves along the pH scale depending on the nature of the buffer used. l The ion exchange selectivity can be influenced by adding organic modifiers such as methanol, acetonitrile or tetrahydrofuran to the eluent. l Alumina is an interesting stationary phase for protein separations, especially in the case of basic proteins. The combination of the size-exclusion and
Fig. 5. Chromatogram of some standard proteins. Mobile phase: 0.25 M Na2HP0, (pH 9). Solutes: 1 and 2 = bovine albumin; 3 = ovalbumin; 4 = myoglobin; 5 = unknown; 6 = arginine vasopressin; 7 = trypsinogen; 8 = lysozyme; 9 = chymotrypsinogen.
the ion-exchange mechanisms allows excellent separations to be obtained by simple means. l Basic solutes can easily be chromatographed on alumina resulting in symmetrical peaks. Columns of high efficiency can be packed in methanol and usually have a high permeability.
References This article is an abstract of Claude Laurent’s Ph.D. thesis entitled: A reappreciation of alumina in high pressure liquid chromatography. The work was done at the laboratory of Analytical Chemistry of the Delft University of Technology under the supervision of Prof Dr L. de Galan. The thesis contains the following papers: 1 C. J. C. M. Laurent, H. A. H. Billiet and L. de Galan, Chromatographia, 17 (1983) 253. (On the use of alumina in HPLC with aqueous mobile phases at extreme PH.). 2 C. J. C. M. Laurent, H. A. H. Billiet and L. de Galan, Chromatographia, 17 (1983) 394. (The use of organic modifiers in ion-exchange chromatography on alumina. The separation of basic drugs.). 3 C. J. C. M. Laurent, H. A. H. Billiet and L. de Galan, J. Chromatogr., 285 (1984) 161-170. (High-performance liquid chromatography of heroin samples on alumina by ion exchange in mixed aqueous-organic mobile phases.). 4 C. J. C. M. Laurent, H. A. H. Billiet, L. de Galan, F. A. Buytenhuys and F. P. B. van der Maeden, J. Chromatogr., 287 (1984) 45-54. (High-performance liquid chromatography of proteins on alumina.). H. Billiet, C. Laurent and L. de Galan are at the Laboratorium voor Analytische Scheikunde, Technische Hogeschool Delft, Jaffalaan 9,2628 BX Delft, The Netherlands.