Analytical and Preparative Applications of Liq uid- LiqIJid Pa rti t io n Chroma to gra phy S. M. LAMBERT and P. 5. PORTER Shell Development Co,, Modesto, Calif.
b With increasing emphasis placed on separation and identification o f newly synthesized technical chemicals, liquid-liquid partition chromatography (LLC) has proved itself an invaluable analytical and preparative tool. Compounds which decompose in gasliquid chromatographic (GLC) systems or are difficult to separate by GLC because of unfavorable vapor pressure relationships are arnenable to LLC. Techniques and equipment for liquidliquid chromatography have been developed which close!ly parallel those used with GLC apparatus currently available. The LLC operation utilizes a pressurized flow scheme incorporating an automatic reco-ding differential refractometer as the, detector. For preparative applications, the system is coupled to an automatic fraction collector and solenoid operated funnel arrangement. A nofel sample injector has been desigied which facilitates the addition of samples to the column without release of pressure. The injector incorporales a micrometer syringe assembly with which small volumes may be delivered with good precision. Preparative quantities of a number of technical chemicals have been obtained by I.LC for use as analytical standards. In addition, LLC has been used for component identification, for quantitative analysis, and for determination of highly accurate partition coefficients for use in molecular structure correlcitions. A computer program for resolution of chromatographic elution curves into Gaussian bands which compose them has been applied successfully to LLC recorder profiles.
C
is perhaps the most widely used method of molecular separation for all types of compounds. Techniques and equipment for liquid-liquid chromatography (LLC) have not progressed as rapidly as those of gas-liquid chromatography perhaps because until recently a stable, compact, universal, high sensitivity detector was not available. The authors feel, however, that L>LC provides a valuable adjunct to GLC techniques, and have attempted to develop techHRORZATOGRAPHY
niques and equipment for LLC operation which closely parallel GLC apparatus currently available. Vnndenheuvel and Sipos (12) have recently described an improved liquid elution column system primarily designed for the comparative study of packing materials. AIany compounds which decompose on GLC systems or are difficult to separate by GLC because of unfavorable vapor pressure relationships are amenable to LLC. Among the first class are compounds which are thermally unstable, those which are subject to catalytic degradation, and those which decompose because of substrate interaction. Among the second class are cis and trans isomers. In liquid-liquid partition chromatography a separation is effected by distribution of the components of a mixture betn een a stationary liquid phace and a mobile liquid phase. Using a column, the mobile phase is allowed to percolate through a packed granular material which acts as a support for the stationary phase. Those components of the mixture which partition more into the stationary phase are retarded with respect to those which partition more into the mobile phase. As opposed to classical adsorption chromatography with its inherent difficulties due to nonlinearity of adsorption isotherms and the ease of deactivation of adsorbents, partition chromatography with its linear isotherms is more amenable to theoretical interpretation and is more reproducible. The superiority of partition chromatography is clearly evidenced by the widespread use of gas-liquid chromatography, paper-partition chromatography and even countercurrent extraction type apparatus (Craig machines). The theory of partition chromatography has been discussed by Martin and Synge ( 7 ) , Klinkenberg and Lapidus Sjenitzer ( 5 ) , Keulemans (4, and Amundson (6), and others (1, 3, 9, 11). Various simplifying assumptions are usually introduced into chromatographic theory to obtain basic differential equations which yield themselves t o mathematical solution. For liquid-liquid partition chromatography the assumption of a linear partition isotherm is usually a good approsimation. Van Deemter, Zuiderweg, and Klinkenberg ( 2 ) have discussed the
mechanisms of band broadening in linear, nonideal chromatography. A Poisson distribution function is derived which is sufficient for calculating the position and shape of a band moving down the column. If the number of theoretical plates is large, this function may be approximated by a Gaussiantype distribution. Martin and Synge (7) have provided a useful expression for the concentration of a solute, C,, in the effluent, as a function of the volume of mobile phase, VI passed through the column. This expression can be reduced to
where r, is the number of theoretical plates in the column for component i, q, is the amount of component i charged to the column, and V E is the retention volume of component i. The retention volume, V R , for a component i s related to the partition coefficient, K,, of the component by the following equation : V R= V m
+ KsV,
(2)
where V , is the volume of the column occupied by the mobile phase and V , k the volume occupied by the stationary phase. K , is defined as the equilibrium ratio of the concentration of solute in stationary phase to that in the mobile phase expressed in suitable units. There are, in addition, two equations which are useful in determining column performance; the number of theoretical plates, T , is found by the relation: ri
= 16
($)'
(3)
where W is the width of the peak a t the base. An expression for the resolution, R, of two components i and j is given by R =2
(w,sj) (4)
PRACTICAL CONSIDERATIONS
Selection of solvent pairs, stationary phase support, and column dimensions are dependent upon the objectives desired. However, the most important factor to consider is the need to obtain stable, compatible, two-phase solvent system-support combinations. One arbitrary test of system stability is VOL 36, NO. 1, JANUARY 1964
99
__
TO
7 +
d
VFNT
1
RECORDER
FRACTION
COLLECTOR
Figure 1.
Block diagram of LLC system
under pressures of 2 to 20 p.s.i.g. The cam is equipped with a micrometer setting complete with vernier and scale for adjustment and reproducibility of A ow rates. Rates are continuously adjustable from 15 to 750 ml. per hour with a l/g-inch bellows. With a high sensitivity detector-e.g., the Waters Associates differential refractometer-it is necessary to damp out the pulsations of the pump. This was accomplished by inserting a stainless steel, adjustable, spring-loaded bellows as a "tee" into the line immediately downstream from the pump. This arrangement, together with a throttling valve placed between the bellows and column inlet, produced a smooth and conqtant flow to the detector. SAMPLEINJECTOR -~PPARATUS. Conventional methods of adding samples to liquid chromatographic columns involve permitting the level of mobile phase in the column to reach the top of the column packing and then pipetting the desired quantity of material onto the packing, avoiding disturbance as much as possible. The sample level is allowed to drop to the top of the packing, mobile phase is added in smaIl quantities to wash down any residual sample on the column walls, and finally the column is refilled with mobile phase and repressurized. Flow rates usually need some readjustment and during the repressurizing step entrapped air may dissolve in the mobile phase. A bubble trap or a flow system which allows the release of pressure downstream from the detector cell is necessary to prevent bubbles from interfering with the detector. This procedure for adding sample is very inefficient and leads to other difficulties in addition to the air bubble.. Pipetting of volatile solvents is difficult and not too reproducible; release of pressure and repressurizing will change the base line of a sensitive detector system such as the differential refractometer; and the possibility always exist. that the top of the column packing will go dry in which case the chromatography cannot be continued. To overcome these difficulties, a sample injection system, Figure 2 , has been constructed which
stationary phase before use. The particle size distribution of the solid support should be uniform, allow good flow characteristics, and prevent channeling. A particle size of 100 to 150 microns is usually satisfactory. If the differences in the partition coefficients of the components to be separated are small, a longer column will be needed to achieve a separation. For preparative chromatography, since one desires a greater capacity, the crosssectional area should be large. whereas a small cross section is desirable for analytical purposes. Of course high purity solvents should be used and if evaporative procedures are required for recovery of sample components, as for preparative work, the mobile phase should be relatively volatile. EXPERIMENTAL
negligible change in retention volume of standard samples injected periodically over a 2- to 3-week period. Experience has shown that if the columns are stable for 2 to 3 weeks they will remain so indefinitely, providing they are not subjected to abnormal stress. For best analytical results the solvent system chosen should have a low mutual solubility and the mutual physical properties of the solvents should be relatively insensitive to temperature changes (thus eliminating many multi-component systems). For convenience, experience has shown that the sample should have an average partition coefficient such that RV. is approximately equal to Vm-i.e., for an effective K V p - 1 where the volume of mobile phase in the column is five times that of the stationary phase-the average partition coefficient of the sample should lie close to 5 . The stationary phase support should be an inert, stable, and porous granular solid which can hold over 50% of its dry weight of the polar stationary phase. The stationary phase should be sorbed strongly on the support while the mobile phase and the solutes to be separated are not. Sorption of the polar phase on the support cannot be overemphasized if one is to achieve a stable column. While many two-phase systems may give the desired partition values, these phases must be applicable to the chosen support. In some cases the use of a surface conditioner or "anchor" may render a support more compatible with the desired stationary phase. For example, a small amount of water mill increase the stability of some alcohols on Celite type supports. A backlog of experience with various solvent systems and stationary phase supports will allow one greater flexibility in choosing proper conditions for desired separations. The mobile phase chosen for any system must, of course, be equilibrated with the 100
ANALYTICAL CHEMISTRY
Apparatus and Technique. The following techniques and equipment which will be described have been used successfully for over 4 years on both the preparative and analytical scale. Figure 1 is a block diagram of the system. Teflon, glass, and stainless steel are the only materials of construction allowed t o contact the mobile phase. COLUMNS.Because of problems of corrosion encountered with metal tubing, industrial glass pipe columns have been used almost exclusively. Fischer & Porter glass pipe, glass fittings, and couplings are available from l/r-inch to I-inch i.d. For chromatographic applications requiring long path lengths, the standard glass pipe columns can be arranged in series with U-bend connectors. The columns which have been used for preparative applications are 3/4-in~hi.d. while those which have been used for analytical work are inch i.d. PUMPS.A microbellows pump consisting of three units, an explosion proof motor, Boston gear box, and an adjustable cam and bellows arrangement delivers the mobile phase t o the column
-LI
i iNJEC-OII
,
YALYE LSSEHBL" ,SI
T'"-.r
Figure 2.
*.ET
WJEC-CR DISPLACEMENT P S 7 i h 5 ML c I P P : , i "
A i
Sample injector apparatus for liquid-liquid chromatography modified design
lox 0
I
m
/A++enuation
a t 90% F ~ I SI c a l e
m
I
li\
Sample Injection
I1
-
0
Sp'ke
Return t o I X /
-
I
J
-Time
Figure 3. LLC profile representing analytical separation of cis-fransisomers of an organophosphorus cornpound
facilitates the addition of variable size samples to the column without release of pressure or change in flow rate. The system is composed of two component parts: ( A ) injector displacement assembly and, (€3) injector valve assembly. Part A shown in Figure 2 is composed of a metric micrometer, piston assembly, cylinder head, zero adjusting stop, and necessary clamping arrangements. Part B as shown is made up of a spring-loaded valve assembly with ports provided for the mobile phase and the sample material. Two microvalves are provided which are used when obtaining a hydrxtatic system on the injector side of thct valve assembly. Once the system on the injector side is purged of air and made hydrostatic, the desired volume is delivered simply by closing valves 1 arid 2 and turning the micrometer the correct number of turns. The piston is constructed so that each micrometel division corresponds to 0.0020 cc. The completely assembled system was tested for repeatability and showed a maximum standard deviation of the mean of &0.004 ml. over the range 0.08 to 1.30 ml. The columns are never depressurized, successive samples can be
injected a t any time interval desired and the columns can never run dry, Samples in the range of 0.05 to 5.00 ml. can be injected with good precision, This injector system offers an advantage over a multiport switching valve in which sample loops must be changed for accurate injections of different volumes. DETECTORS. The main detectors used in this work were two Phoenix automatic recording differential refractometers having a sensitivity of 1.4 x Refractive Index Units (R.I.U.) and a Waters Associates differential recording refractometer having a sensitivity of 7 X lo-* R.I.U. Both refractive index and ultraviolet absorption detectors have been employed for continuous monitoring of column effluents. In general, the differential recording refractometer is preferred because of its versatility and ease of operation. PACKING OPERATION. Besides the advantages of having a closed system using constant-volume pumps and precision sample injectors, a pressurized LLC system offers an expedient solution to column packing. The columns may be packed dry-i.e., with no mobile phase present but with the solid support
containing the sorbed stationary phase -and then degassed by simply pumping the mobile phase through under pressure (20 to 30 p.s.i.g.). Most of the air is displaced by the incoming liquid and that which remains dissolves under pressure in the mobile phase. For an 8-foot by 3/4-in~h i.d. column, this degassing operation takes about 40 minutes after which the column is ready for operation. Packing columns arranged in series requires the use of some support to keep the columns positioned rigidly A during the packing operation. wooden packing frame has been adopted for this purpose. The columns are packed individually and in reverse order. The last column of the series is packed first after which the U-bend is connected, the column inverted, and the U-bend packed. The next column is then connected and packed, repeating the procedure for each successive column. A length of Teflon tubing with the ends sealed by cork stoppers serves as a tamping device for packing around the U-bends. The completed assembly is finally righted and fastened to the supports with the packing frame in place. After the columns are adequately supported by clamps, the copper wire fasteners which hold the columns to the frame are cut and the frame is removed. Care must be exercised to assure correct alignment. FRACTION COLLECTOR.Since preparative chroniatographic separations on long columns consume a great deal of time and since most of the effluent is pure solvent, the differential refractometer was modified for automatic operation with a preparative model fraction collector. The refractometer recorder was adapted to activate the fraction collector only when components were emerging from the column. An adjustable cam and microswitch attached to the recorder potentiometer activates a relay system when the recorder moves off the base line. This relay activates the fraction collector and a solenoid operated funnel arrangement to divert the effluent into a central collecting tube on the fraction collector. As the recorder pen describes the chromatographic curve, fractions are collected a t preset intervals. When the pen returns to the base line, a timer is activated which allows a preset safety period to elapse before the collector is deactivated. MThen the collector is deactivated, the funnel arrangement directs the effluent to a discard reservoir. Each time the pen moves off the base line the safety timer is automatically reset and the fraction collector reactivated. Using this detector-collector system, time consuming chromatographic runs can be made with the equipment essentially unattended. SOLVENT RESERVOIRS.The cabinets in the upper portion of Figure 1 are used to hold the solvent reservoirs which are 2I/~gallonborosilicate glass carboys. The cabinets are vented through a laboratory hood and equipped with a spring-loaded damper arrangement with a fusible trigger to isolate each reservoir VOL. 36, NO. 1, JANUARY 1964
101
independently in case of fire. Pressure relief valves, pressure gauges, and filter systems comprise the rest of the system.
I
RESULTS
Figure 3 is a typical chromatographic profile obtained using an analytical apparatus. It represents the cis-trans isomer separation of an organophosphorus insecticide. The time required for the separation wm less than 1 hour. This may be contrasted with typical chromatographic profiles obtained during preparative separations, Figures 4 and 5, which represent the cis-trans isomer separation for two commercial insecticides, Phosdrin insectiride and Ciodrin insecticide. Peaks which fall below thp hase line indicate the presence of components having rrfractive indices lower than that of the reference material, normally the mohile phase. Technical Phosdrin insecticide, Figure 4, is composed of two isomers and a minor impurity arising from the method used for synthesis. Technical Phosdrin Insecticide
0
CHaO ‘P7
C H k ‘0
H
’OCII,
cis-crotonate derivative CHs H
0 m
0
I\
0
W
LT
a I
0
m
I \WA
0 N
a hexane-CC14 solution as the mobile phase and a HeO-ethylene glycol solution as the stationary phase. The stationary phase support was C-22 Silocelfirebrick 100 to 120 mesh. HETP for these preparative columns range from 0.1 to 0.2 inch. The columns are quite stable, some being in use for over 4 years with negligible changes in retention volumes for standard samples. LLC may be used for both qualitative and quantitative analysis. In LLC, uaing a differential refractive index detector, the ares under the corresponding peak, is proportional to the amount of that compound present. While it is true that detector response using RI varies for different materials, LLC may be used to compare relative amounts of
CH,.0’ tram-crotonate derivative
OD
I
OCH, chloro impurity
102
ANALYTICAL CHEMISTRY
individual components; and, in cases where pure standards have been separated, calibration is readily accomplished. For quantitative and qualitative aspects of LLC using the differential refractometer detector, the higher the sensitivity of the detector the more freedom one has as to choice of solvent systems. One system used for analytical purposes consists of an automatic recording differential refractometer used in conjunction with a 1I4-inch i.d. column. Introduction of 100 11. of a 0.10% solution of a compound having a refractive index of ca. 1.50 (solvent refractive index = 1.38) will produce a peak having a height of 30% full scale under conditions where the noise level is under 1%. Figures 6
0 01
0
The first peak (negative detector displacement) is the chloro material and amounts to ca. 2.5% while the second and third peaks (positive detector displacement) 5re the cis and trans isomers, respectively. In general, preparative separations require 3 to 5 hours and will produce 1OOIto 1000 mg. of pure material using %foot X */4-inch i.d. columns. Determinations of purity are confirmed by a number of independent techniques consisting of calorimetry, XMR, infrared, elemental analysis, and GLC where applicable. The separations shown in Figures 4 and 5 were accomplished using
\
I I :
Q
I
0
1
is particularly valuable for liquidliquid chromatograms because of the close conformance of the bands to the Gaussian shape. In applying the computer program to elution curves for Ciodrin insecticide, it was found that the peak which had been assumed to represent only the cis-crotonate isomer was composed of two peaks lying very close together. This was not discernible from visual examination and was not readily evident from measurements made with ruler and compass. To verify the presence of two peaks, Ciodrin was again chromatographed using a column of twice the length and with lower flow rate to achieve higher resolution. The chromatogram now showed the two components as indicated by the computer while fractions corresponding to both components were
Partition Coefficients and Molecular Structure. LLC provides a means of obtaining highly accurate values for partition coefficients without the need for having pure samples. These accurate partition coefficients in turn provide a powerful tool in structure confirmation. The relationship of activity coefficient (or partition coefficient) to molecular structure has been aptly described and experimentally verified by Pierotti, Deal. and Derr (8). These authors 5how that the logarithms of activity coefficients for members of solute families a t infinite dilution fall into simple correlation patterns which are in turn susceptible to a simple, semiempirical interpretation based on molecular interaction. "For methylene hmiologs, RIXI, a t infinite dilution in methylene homologs, RPXrtJthe pattern ib described by: FIE/2.3RT= log C l h D(nl
+
Figure 6. Typical L1.C profile obtained using analytical appsaratus
and 7 show typical chromatographic profiles obtained tvith this apparatus. The average analysis requires less than 1 hour. These columns have plate heights ranging from 0.05 to 0.10 inch. To increase the sensitivity and stability of this unit, the columns, solvent systems, and detector have been assembled in a carefully controlled thermostatted cabinel;. Liquid-liquid chromatography has been used to compiire the technical materials produced under varying reaction conditions acd with different routes of synthesis. Even in cases Khere preparative cliromatogrsphy is not the primary objec tive, the material corresponding to recorder peaks may be collected and run quaatitatively in the infrared using a mi1:rocell technique. This procedure is quite useful for the identification of impurities present in small quantities in the original sample. Computer Program for Data Reduction. Since complete resolution of bands during liquid-liquid chromatography is not always achieved, i t is sometimes difficult i o determine the amount of a component present in the sample. The various bands emerging from the column are symmetrical and can be described mrithematically by Gaussian distribution functions so that by successive approximations and with much measuring and drawing the detector curve can be resolved into the individual curves aliich compose it. Stone (IO) of the Shell Development Co., E m e r y ille Research Center. developed a computer program which is capable of resolving chromatographic elution curves into the Gaussian bands which compose them. This resolution
= A1,*
-
nzY
+ B2nl/nn+
+F
h (5)
where
-FIE
4
partial molal free energy of solute a t infinite dilution in the solvent in excess over the Raoult's law value (pure liquid a t its saturation pressure as standard state). yto = activity coefficient of component RIXl a t infinite dilution in component R2X1. n,,nz = numbers of carbon atoms in hydrocarbon radicals RI and R2, respectively. A1,2 = coefficient which depends on nature of solute and solvent functional groups, X1 and X2. BQ = coefficient which deuends only on nature of sdlvent functional group, XI. C1 = coefficient which depends only on solute functional group, x1 D = coefficient independent of both X1 and X,. =
Figure 7. Typical LLC profile obtained using analytical apparatus
collected and characterized by infrared techniques. The detector curve, part of which is shown in Figure 8, was composed of si. components, some of which were not resolved and which produced both positive and negative refractive index changes m-ith respect t o the reference solvent. The residual curve for the observed minus calculated profile is within limits of experimental error a t all points. Figure 8 shows the detector envelope, solid line, and the components found by the computer, broken lines. Once the detector envelope is resolved, it becomes a simple matter to determine the amount of each component present in the original sample.
Figure 8. Computer resolution technical Ciodrin insecticide VOL. 36, NO. 1, JANUARY 1964
0
for 103
Fg
= coefficient which essentially
depends only on nature of the solvent functional group, X X . “For simple interpretation it is assumed that excess free energy or log y o can be treated as a sum of contributions from individual interactions between pairs of structural groups in the solute and solvent molecules which depend solely on the number, type, and configuration of the groups in the respective structures.” The relationship between partition coefficients and molecular structure is evident from retention volume data obtained by LLC. Choosing one twophase system as a standard, one finds that relatively constant differences in
log VOR (corrected retention volumes) are associated with changes in molecular structure for related compounds. This information enables prediction of log VORfor structure confirmation of newly synthesized compounds. In practice, Alog VOEdata have been used to predict the configuration of newly synthesized compounds with excellent results. LITERATURE CITED
( 1 ) Cassidy, H. G;! “Fundamentals of
Chromatography, Vol. X, Interscience, New York, 1958. (2) Deemter, J. J. van, Zuiderweg, F. J., Khkenberg, A,, Chem. Eng. Sci. 5, 271
(19.5Rl (3) De Vault, D., J . A m . Chem. SOC.65, 532 (1943). (4) Keulemans, A. I. M., “Gas Chromatography,” Reinhold, New York, 1959. \ - - - - I -
(5) Klinkenberg, A,, Sjenitzer, F., Chem. Ena. Sci. 5.258- f 1956’i. (6) LLpidus, L., Amundson, N. R., J . Phys. Chem. 56,984 (1952). I
\ - - - - ,
( 7 ) Martin, A. J. P., Synge, R. L. M., Biochem. J . 35, 1358 (1941). ( 8 ) Pierotti, G. J., Deal, C. H., Derr.’ E. L., Ind. Eng. Chem. 51,95 (1959). (9) Porter, P. E., Deal, C. H., Stross, F. H., J . Am. Chem. SOC.78, 2999 (1956). (IO) Stone, H., J . Opt. SOC.Am. 52, 998 (1962).
R,;, “Column Partition Chromatography, Merck and Co., New Jersey, 1957. (12) Vandenheuvel, F. A., Sipos, J. C., J . Chromatog. 10,131 (1963). (11) Trenner, ru’.
RECEIVEDfor review May 8, 1963. Accepted October 23, 1963. Presented in part, a t the Pittsburgh Conference on
Analytical Chemistry and Applied Spectroscopy, March 1963.
Resolution of Neomycin and Catenulin Antibiotic Complexes by Ion Exchange Resin Chromatography HUBERT MAEHR and CARL P. SCHAFFNER Institute o f Microbiology, Rufgers, The Sfate University, New Brunswick, N. 1.
b The neomycin antibiotic complex (neomycin A or neamine, neomycin B, and neomycin C) has been separated b y ion exchange resin column chromatography. The procedure involves the use of a strongly basic anion exchange resin of low cross linkage, Dowex 1-X2, in the hydroxyl form and water as the eluent in a one-step elution operation. Application of this technique to the structurally closely related catenulin antibiotic complex resulted in the detection of four components, two obtained as pure compounds and two obtained in mixture. This chromatographic technique can be applied either for analysis or in large-scale preparative operations.
N
(23) and catenulin (3) are typical water-soluble, basic antibiotic substances that are produced by Streptomycetes as complexes of closely related or even isomeric components. The neomycin complex is composed of three antibiotic substances, neomycin A (neamine) ( 2 1 ) and the isomeric neomycin B and neomycin C (4, 6). Neamine is a biologically active structural moiety of neomycins B and C. The catenuliin complex was thought to consist of three components, as suggested by paper chromatographic studies with the N-acetyl derivatives (20, 21). The complex of catenulin was believed to be similar, if not identical, to that of paromomycin ( 7 ) , hydroxymy-
104
EOMYCIN
ANALYTICAL CHEMISTRY
cin (6),and aminosidin (1). The identity of these complexes has been verified in recent studies (21). Recently the structures of two components of the paromomycin complex have been compared with those of the closely related neomycins B and C (16). Japanese investigators (9, 10) likewise report structures of two components of the zygomycin A complex (8), revealing their identity to the two components of the paromomycin complex. In the past a great deal of research effort was directed toward the difficult problem of separating the neomycin antibiotics. Countercurrent distribution studies (19, $2) indicated the inhomogeneity of neomycin preparations. Separations obtained with this technique were a t best poor as well as unsatisfactory for the determination of minor components. In 1951, Dutcher, Hosansky, Donin, and Wintersteiner (4) described the first separation of neomycins B and C by alumina column chromatography, employing the hydrochlorides and 80% aqueous methanol. I n the purification of crude neomycin concentrates by carbon chromatography (6) neomycins B and C were partially separated. Crude neomycin sulfate preparations as concentrated aqueous solutions were added to a column packed with an aqueous slurry of acidwashed Darco G-60 and kieselguhr. With aqueous development a t pH 2, most inorganic salts passed rapidly through the column and were closely followed by neomycin C-rich fractions.
After overlapping fractions of combined neomycins B and C, fractions rich in neomycin B were obtained. With the extensive overlapping of components even rechromatography gave incomplete separation of the antibiotics. More recently a carbon chromatographic method for the resolution and quantitation of neomycin antibiotic complexes was introduced (8). This method is based on the separation of neomycin sulfates by thin-layer chromatography employing carbon black. The biological activity on the thin layer plates is detected by an agar diffusion technique. Attempts a t the paper chromatographic separation of neomycin complex preparations generally were unsuccessful. Differentiation of purified preparations of neomycins A, B, and C was reported by Saito and Schaffner (17). Whereas the complex could not be resolved completely, individual neomycin components demonstrated different migrations on paper saturated with 1.OM sodium sulfate, using aqueous saline-methanol as the developing solvent. Pan and Dutcher (IS) effectively separated and differentiated preparations of neomycins B and C by paper chromatography of their N-acetyl derivatives. Preparative separations by liquid-liquid partition chromatography of the N-acetates on cellulose columns have also been reported (14). Since the N-acetyl neomycin derivatives are biologically inactive, these methods have only analytical interest.
