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Simple, multibore columns for superior fractionation of lipids
ANALYTICAL
Simple,
BIOCHEMISTRY
9, 303-309
Multibore
(1964)
Columns
for
of GEORGE From
the Biochemistry
Superior
Fractionation
Lipids1
A. FISCHER
JON J. KABARA
AND
Research Division,
University
Received February
of Detroit,
Detroit,
Michigan
26, 1964
Studies involving the use of various solvent systems and adsorbents have occupied investigators concerned with improving the efficiency of column chromatography (l-3). Throughout these studies, the role of column design has been the least appreciated variable. In order to achieve better resolution with increased sample size, the effects of column bore, length, and their relative geometries were investigated. The study of column design presented herein is limited to the separation of the neutral lipids on Florisil. The principles involved, however, are applicable to other systems. METHODS
All the different columns studied had internal volumes of 15 cc and contained identical amounts of adsorbent. Temperature was maintained between 22 and 24°C in an air-conditioned room. In all cases, flow rates TABLE SOLVENT
SEQUENCE
AND
VOLUME
Flow rates can be doubled without Solution
I II III IV V VI VII VIII Total Time
Hexane 5% ether in hexane 15y0 ether in hexane 25y0 ether in hexane 50% ether in hexane 2% MeOH in ether 4% HOAc in ether MeOH volume at 0.5 ml/min
USED
1 IN
SEPARATIONS
REPORTED
greatly influencing the separation. Cd.
A
20 50 70 70 50 50 50 90 450 15
ml ml ml ml ml ml ml ml ml hr
Cd.
B
20 ml 35 ml 45 ml 50 ml 35 ml 35 ml 35 ml 75 ml 330 ml 11 hr
Cd.
c
20 30 35 40 30 30 30 65 275 9
ml ml ml ml ml ml ml ml ml hr
Col. D
20 30 30 35 30 30 30 50 250 8
ml ml ml ml ml ml ml ml ml hr
‘This research was supported in part by the National Institute of Health, Division of Neurological Disease and Blindness (NB-02239, Michigan Heart Association, and Michigan Multiple Sclerosis Society. 303
304
I:ISCHER
AiYD
KABARA
were adjusted to 0.5 ml/min. The eluents were collected in 2-ml fractions, on an automatic drop-counting fraction collect’or. Essentially, the procedure for the separation of neutral lipids was that employed by Carroll (4). The Florisil used for fractionation was 60-100 mesh, and hydrated to contain 75% water. The sequence of addition and optimum solvent volumes was experimentally determined for each column as illustrated in Table 1. The column load, at all times, was approximately 40 mg of lipid extracted from mouse liver. Analyses were made of the various fractions using the following methods: hydrocarbons by gas chromatography ; cholesterol and cholesterol esters by the Lieberman-Burchard
24140 Joint
Zx130mm
24/40 Joint
24/40 Joint
1
24/‘40 Joint
iX600mm
Needle ValW
6xl50mm
.25xl50mm
3X150mm
Needle VZIlVfA
1. Representative Long narrow-bore
FIG.
(B)
Needle valve
column designs: (A) column column. (C) Two-bore column.
, Needle W.lW
described hy (II) Four-bore
Ii.
B. Carroll, column.
reaction (5) ; and glycerides by the method of Korn (6j. In addition, all fractions were checked for homogeneity by thin-layer chromatography. The data for each column in t,he accompanying figures were tabulated by averaging values from ten to twenty experiments. RESULTS
Straight Columns A number of st,raight-bore columns of varying lengths but identical volume (15 cc) were used to determine the effect of elongation on separation. Two extreme casesof straight column design are shown as A and B in Fig. 1. Graphically, the separations on these columns are represented in Figs. 2 and 3, respectively. Data from this series of columns showed
50 ml loom, 200 ml 50% 250 Ether 256 Ether Ethert150nll 5% Ethert 15Gi t FIG. 2. Separation
of neutral
lipids on 12 x 130 mm column C-4 in Fig. 1).
several effects: as column length increases, separation (distance between solute peaks) increases; as column bore decreases, resolution (shape of solute peaks) increases; capacity and the amount of the elution solvent required increase as function of the square of t,lre radius.
FISCHER
FIG.
3. Separation
of neutral
AND
I
lipids on 6 x 600 mm column (B in Fig. 1).
Multibore
Columns
The undesired effect of lower capacity when the bore is decreased in a straight column can be seen to be interfering with the desired effect of increased resolution when the same load is applied to both columns. In low-capacity applications (<5 mg of total lipid) a very satisfactory compromise can usually be achieved. When greater amounts of material (>5 mg of total lipid) are to be separated on single bore columns, no acceptable compromise can be reached. In this case, large amounts of adsorbent and extremely large solvent volumes are required in order to achieve good separation. In order, therefore, to achieve high capacity with good resolution and reasonable elution volumes in a single column, it was necessary to think in terms of a multibore column. The theory regarding columns with diminishing bores was evolved by Hagdahl (7, 8)) who reported success with such coupled columns. However, the application of the technique utilizing composite columns has not found widespread use. Technical difficulties arise in packing columns, separated by capillary tubing, which lead to the formation of bubbles and air pockets. The past difficulties of using such multibore, coupled columns were
MULTIBORE
Fm. 4. Separation
Column
3Oi
COLUMN8
of neutral lipids on %-bore column (C in Fig. 1).
‘TV
t
1 kkxane 1 5% Ether
1 15% E&P
FIG. 5. Separation
1
25% Ether
1 56 Ether t
22:
1 z,‘z
of neutral lipids on 4-bore column
1
Mem
(D in Fig. 1).
308
FISCHER
AND
KhBARA
overcome simply by joining glass tubing of various diameters directly, without a capillary intermediate. Coupled columns of this type had greater capacity and gave greater resolution than did conventional single-bore columns. An ideal ratio between the two-bore radii was found essential for optimum separation and resolution. Empirically, a satisfactory ratio was found to be where the relationship of the upper to lower radius was 1.4:1, where the two sections were equal in length. A column of this design is sketched in C in Fig. 1. The separations are represented graphically in Fig. 4. Further extension of this principle with columns of three and four sections gave even quicker and better resolutions of the lipid extract (Fig. 5). DISCUSSIOPlT
The effect of bore radius on column capacity is not difficult to understand. A certain cross-sect’ional area of adsorbent. is necessary to adsorb a given amount of solute. The effect of length on separation also requires little comment. The more exchange that takes place in a column, the farther a faster moving solute will be separated from a slower moving one. The effect of the bore on amount of solvent required also can be interpreted. In a longer, thinner column, each solvent molecule contacts more adsorbent particles, thus increasing the efficiency of each molecule of solvent. A further beneficial effect of the column bore results from the reduced influence of tilt and channeling in a narrower column. Under usual conditions, column chromatographic separation of a complex mixture results in irregular zone fronts down the length of a single long column. To obviate this fault, Hagdahl (7, 8)) Claesson (9), and Pilley et al. (10) recommended the use of “divided columns.” These recommendations improved the sharpness of separation. Technical difficulty in preparing this type of column for routine use has discouraged its widespread adoption, This difficulty was overcome by connecting a series of columns of diminishing radii. The results were multibore columns of high capacity and superior resolution. The effect of column length and radius should be kept in mind when interpreting the success of multibored columns. An initial large cross section of adsorbent is necessary for high capacity. This need is met in the first section. Initial alignment of the solutes also is achieved in this first section, in spite of the degree of tilt and channeling. In each succeeding section, the form of the solute bands improves, due to the smaller bores in the lower sections. This is accompanied by increased linear separation in each section. The emerging band of solute is as sharp as that from a column with a bore equivalent to t,hat of the smallest section. The
XICLTIBORE
309
COLIXXS
capacity is almost equivalent to that of a column with a bore equal to that of the top section. Thus, columns giving superior fractionation and high capacity can be constructed, using the principle of multibores. SUMMARY
Multibore columns were constructed and found to have a high capacity, give sharp resolution, with less adsorbent and smaller amounts of eluting solvent. A separation requiring 12 hr on a straight column can be accomplished in one-third less time on a multibore column. This factor, added to the superiority of fractionations on multibore columns, has proved to be of great benefit in routine analysis. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
HOLMAN, R. T., J. Am. Chem. Sot. 73, 1261, 3337, 5289 (1951). TRAPPE, W., Biochem. J. 306, 316 (1940). KLEIN, P. D., AND JANSSEN, E. T., J. Biol. Chem. 234, 1417 (1959). CARROLL, K. K., /. Lipid Res. 1, 171 (1960). SPERRY, W. M., AND WEBB, M., J. Biol. Chem. 187, 97 (1950). KORN, E. D., J. Biol. Chem. 215, 1 (1955). HAGDAHL, L., Acta Chem. Scnnd. 2, 574 (1948). HAGDAHL, L., LKB Instrument J. 1, T&o. 3, 21 (1954). CLAESSON, S., Arkiv Kemi Mineral Geol. 16, 24A (1957). PILLAY, P. P., RAO, D. S., N.4IR, C. P. N., AND VORKEY, E. T., Chem. No. 9, 258 (1958).
Ind.
London
Simple,
BIOCHEMISTRY
9, 303-309
Multibore
(1964)
Columns
for
of GEORGE From
the Biochemistry
Superior
Fractionation
Lipids1
A. FISCHER
JON J. KABARA
AND
Research Division,
University
Received February
of Detroit,
Detroit,
Michigan
26, 1964
Studies involving the use of various solvent systems and adsorbents have occupied investigators concerned with improving the efficiency of column chromatography (l-3). Throughout these studies, the role of column design has been the least appreciated variable. In order to achieve better resolution with increased sample size, the effects of column bore, length, and their relative geometries were investigated. The study of column design presented herein is limited to the separation of the neutral lipids on Florisil. The principles involved, however, are applicable to other systems. METHODS
All the different columns studied had internal volumes of 15 cc and contained identical amounts of adsorbent. Temperature was maintained between 22 and 24°C in an air-conditioned room. In all cases, flow rates TABLE SOLVENT
SEQUENCE
AND
VOLUME
Flow rates can be doubled without Solution
I II III IV V VI VII VIII Total Time
Hexane 5% ether in hexane 15y0 ether in hexane 25y0 ether in hexane 50% ether in hexane 2% MeOH in ether 4% HOAc in ether MeOH volume at 0.5 ml/min
USED
1 IN
SEPARATIONS
REPORTED
greatly influencing the separation. Cd.
A
20 50 70 70 50 50 50 90 450 15
ml ml ml ml ml ml ml ml ml hr
Cd.
B
20 ml 35 ml 45 ml 50 ml 35 ml 35 ml 35 ml 75 ml 330 ml 11 hr
Cd.
c
20 30 35 40 30 30 30 65 275 9
ml ml ml ml ml ml ml ml ml hr
Col. D
20 30 30 35 30 30 30 50 250 8
ml ml ml ml ml ml ml ml ml hr
‘This research was supported in part by the National Institute of Health, Division of Neurological Disease and Blindness (NB-02239, Michigan Heart Association, and Michigan Multiple Sclerosis Society. 303
304
I:ISCHER
AiYD
KABARA
were adjusted to 0.5 ml/min. The eluents were collected in 2-ml fractions, on an automatic drop-counting fraction collect’or. Essentially, the procedure for the separation of neutral lipids was that employed by Carroll (4). The Florisil used for fractionation was 60-100 mesh, and hydrated to contain 75% water. The sequence of addition and optimum solvent volumes was experimentally determined for each column as illustrated in Table 1. The column load, at all times, was approximately 40 mg of lipid extracted from mouse liver. Analyses were made of the various fractions using the following methods: hydrocarbons by gas chromatography ; cholesterol and cholesterol esters by the Lieberman-Burchard
24140 Joint
Zx130mm
24/40 Joint
24/40 Joint
1
24/‘40 Joint
iX600mm
Needle ValW
6xl50mm
.25xl50mm
3X150mm
Needle VZIlVfA
1. Representative Long narrow-bore
FIG.
(B)
Needle valve
column designs: (A) column column. (C) Two-bore column.
, Needle W.lW
described hy (II) Four-bore
Ii.
B. Carroll, column.
reaction (5) ; and glycerides by the method of Korn (6j. In addition, all fractions were checked for homogeneity by thin-layer chromatography. The data for each column in t,he accompanying figures were tabulated by averaging values from ten to twenty experiments. RESULTS
Straight Columns A number of st,raight-bore columns of varying lengths but identical volume (15 cc) were used to determine the effect of elongation on separation. Two extreme casesof straight column design are shown as A and B in Fig. 1. Graphically, the separations on these columns are represented in Figs. 2 and 3, respectively. Data from this series of columns showed
50 ml loom, 200 ml 50% 250 Ether 256 Ether Ethert150nll 5% Ethert 15Gi t FIG. 2. Separation
of neutral
lipids on 12 x 130 mm column C-4 in Fig. 1).
several effects: as column length increases, separation (distance between solute peaks) increases; as column bore decreases, resolution (shape of solute peaks) increases; capacity and the amount of the elution solvent required increase as function of the square of t,lre radius.
FISCHER
FIG.
3. Separation
of neutral
AND
I
lipids on 6 x 600 mm column (B in Fig. 1).
Multibore
Columns
The undesired effect of lower capacity when the bore is decreased in a straight column can be seen to be interfering with the desired effect of increased resolution when the same load is applied to both columns. In low-capacity applications (<5 mg of total lipid) a very satisfactory compromise can usually be achieved. When greater amounts of material (>5 mg of total lipid) are to be separated on single bore columns, no acceptable compromise can be reached. In this case, large amounts of adsorbent and extremely large solvent volumes are required in order to achieve good separation. In order, therefore, to achieve high capacity with good resolution and reasonable elution volumes in a single column, it was necessary to think in terms of a multibore column. The theory regarding columns with diminishing bores was evolved by Hagdahl (7, 8)) who reported success with such coupled columns. However, the application of the technique utilizing composite columns has not found widespread use. Technical difficulties arise in packing columns, separated by capillary tubing, which lead to the formation of bubbles and air pockets. The past difficulties of using such multibore, coupled columns were
MULTIBORE
Fm. 4. Separation
Column
3Oi
COLUMN8
of neutral lipids on %-bore column (C in Fig. 1).
‘TV
t
1 kkxane 1 5% Ether
1 15% E&P
FIG. 5. Separation
1
25% Ether
1 56 Ether t
22:
1 z,‘z
of neutral lipids on 4-bore column
1
Mem
(D in Fig. 1).
308
FISCHER
AND
KhBARA
overcome simply by joining glass tubing of various diameters directly, without a capillary intermediate. Coupled columns of this type had greater capacity and gave greater resolution than did conventional single-bore columns. An ideal ratio between the two-bore radii was found essential for optimum separation and resolution. Empirically, a satisfactory ratio was found to be where the relationship of the upper to lower radius was 1.4:1, where the two sections were equal in length. A column of this design is sketched in C in Fig. 1. The separations are represented graphically in Fig. 4. Further extension of this principle with columns of three and four sections gave even quicker and better resolutions of the lipid extract (Fig. 5). DISCUSSIOPlT
The effect of bore radius on column capacity is not difficult to understand. A certain cross-sect’ional area of adsorbent. is necessary to adsorb a given amount of solute. The effect of length on separation also requires little comment. The more exchange that takes place in a column, the farther a faster moving solute will be separated from a slower moving one. The effect of the bore on amount of solvent required also can be interpreted. In a longer, thinner column, each solvent molecule contacts more adsorbent particles, thus increasing the efficiency of each molecule of solvent. A further beneficial effect of the column bore results from the reduced influence of tilt and channeling in a narrower column. Under usual conditions, column chromatographic separation of a complex mixture results in irregular zone fronts down the length of a single long column. To obviate this fault, Hagdahl (7, 8)) Claesson (9), and Pilley et al. (10) recommended the use of “divided columns.” These recommendations improved the sharpness of separation. Technical difficulty in preparing this type of column for routine use has discouraged its widespread adoption, This difficulty was overcome by connecting a series of columns of diminishing radii. The results were multibore columns of high capacity and superior resolution. The effect of column length and radius should be kept in mind when interpreting the success of multibored columns. An initial large cross section of adsorbent is necessary for high capacity. This need is met in the first section. Initial alignment of the solutes also is achieved in this first section, in spite of the degree of tilt and channeling. In each succeeding section, the form of the solute bands improves, due to the smaller bores in the lower sections. This is accompanied by increased linear separation in each section. The emerging band of solute is as sharp as that from a column with a bore equivalent to t,hat of the smallest section. The
XICLTIBORE
309
COLIXXS
capacity is almost equivalent to that of a column with a bore equal to that of the top section. Thus, columns giving superior fractionation and high capacity can be constructed, using the principle of multibores. SUMMARY
Multibore columns were constructed and found to have a high capacity, give sharp resolution, with less adsorbent and smaller amounts of eluting solvent. A separation requiring 12 hr on a straight column can be accomplished in one-third less time on a multibore column. This factor, added to the superiority of fractionations on multibore columns, has proved to be of great benefit in routine analysis. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
HOLMAN, R. T., J. Am. Chem. Sot. 73, 1261, 3337, 5289 (1951). TRAPPE, W., Biochem. J. 306, 316 (1940). KLEIN, P. D., AND JANSSEN, E. T., J. Biol. Chem. 234, 1417 (1959). CARROLL, K. K., /. Lipid Res. 1, 171 (1960). SPERRY, W. M., AND WEBB, M., J. Biol. Chem. 187, 97 (1950). KORN, E. D., J. Biol. Chem. 215, 1 (1955). HAGDAHL, L., Acta Chem. Scnnd. 2, 574 (1948). HAGDAHL, L., LKB Instrument J. 1, T&o. 3, 21 (1954). CLAESSON, S., Arkiv Kemi Mineral Geol. 16, 24A (1957). PILLAY, P. P., RAO, D. S., N.4IR, C. P. N., AND VORKEY, E. T., Chem. No. 9, 258 (1958).
Ind.
London