DE930651C - Process for the purification and separation of the B group vitamins by partition chromatography - Google Patents

Description

Process for purifying and separating the vitamins of the Bl2 group by Partition chromatography The two-dimensional partition chromatography of concentrates of vitamin B12 or the B2 group on cellulose is available under the name Papiefchromat, ographie known. W. A. ÄVinsten and E. Eigen (J. biol. Chem. I77, 989; I8I, I09 {1949]) separated vitamin B12 and deoxyriboside using n-butanol as a mobile Phase. H. L. A.

Tarr, B. A. Southcott, and P. W. Ney (Food.

Technol., 4, 354 [I9501) also separated vitamin B12 and deoxyribosides under the same experimental conditions. E. Lester Smith et al.

(German patent 890 265) used paper chromatography resp. the Rf values calculated from the paper chromatograms to distinguish between the vitamins B12, B12b and BI2C- They used water-saturated sec-butyl alcohol as a developer. E. Lester Smith, W.F.J.Cuthbertson, A. Walker, and K. A. Lees (Feder. Proc. 9, No. I [1950]) used higher alcohols or ketones as developers paper chromatographic separation of vitamins B12 and B12b from deoxyribosides. V. Kocher, R. Karrer and H. R. Müller (Intern. Z. Vitaminforschg. 2I, 403 [I950]) dealt with the separation of vitamin B12 and deoxyribosides by paper chromatography using phenol, saturated with an aqueous 2N Na Cl solution or from water-saturated n-butanol or water-saturated col- lidine. H. B. Woodruff and J. C. Foster (J, biol.

Chem. I83, 569 [Ig501) tried by saturation of the paper chromatographic Strip with salt solutions the migration speed of vitamins BI2 and B12 a in the n-butanol-water paper chromatogram. They reached the goal through Use of 0.66 molar K H2 P 04 solutions in which the paper strips are immersed and then dried. The extremely low Rf values of the Vitamin B12 (0.03 to 0, 10) or vitamin B12 a (0.03 to 0.03) can be increased. H. G. Wij menga (dissertation, Utrecht 1951) separated the vitamins B12 and B12 b as well as its BI2 factors WAB, WR and B12 m using n-butanol-water or n-butanol-glacial acetic acid-water with or without the addition of KCN as a developer. Using of n-butanol-water he developed the paper chromatograms for 67 to 72 hours at I6 to I80. The separation effect was extremely low, which is understandable because all vitamin Bl2 factors Wijmengas generally have a lower Rf value as vitamin B12 itself. By using n-butanol-glacial acetic acid-water or by Saturation of the paper strips with KH2PO4 (using n-butanol-water as Developer) the author was able to significantly increase the Rf values. Above the selectivity under these conditions cannot be imagined, since the substances of this author are not uniform according to later investigations was. J. E. Ford, S. K.

Kon and J. W. G. Porter (Biochem. J. 50, Proc. IX [195I]) used paper chromatography for the detection and separation of vitamin B12 factors in faeces and spaces. Using sec-butanol as a developer and in the presence from cyanide they were able to create two new vitamin Bl2 factors, namely factor A (Rf o, I3) and factor B (Rf 0.54) in addition to vitamin B12 (Rf 0.2g) bioautographically with E. Coli prove. M. F.

Patte (from. Pharm. Franc. 9, 747 [I95I]) separated the vitamins B12 and B12 b using n-butanol-glacial acetic acid-water as developer. E. A.

Kaczka, R. G. Denkewalter, A. Holland and K. Folkers (J. Am. Chem. Soc. 73, 335 [I95I]) examined the vitamins B12a and B12a by paper chromatography B1Zb using a mixture of 10% glacial acetic acid, 400/0 n-butanol and 500 / o Water; they found the same Rt value 0.14 for the two vitamins. U. J. Lewis, D. V.

Tappan and CA. Elvehj em (J. biol. Chem. I94, 539; 199, 5I7 [1952]) examined by paper chromatography, using water-saturated n-butanol and water-saturated sec-butanol as a developer, extracts from rat faeces, in who, with the help of this method, obtain a new vitamin Bl2 factor, the vitamin Bl2f, found. E. Lester Smith et al. (Biochem. J. 52, 389 [1952]) give for Whatman-4 paper and sec-butanol-water have the following Rf values: 0.05 for Bl2b, about 0.2 for B12 and about 0.25 for B ,,,. Z. G. Bánhidi and L. E. Ericson (Acta Chem. Scand. 7, 7I3 [1953]) showed paper chromatography using a n-butanol-water-glacial acetic acid mixture as a developer of vitamin B12 b in various algae. A. G. M.

Sjöström and L. E. Ericson (Acta Chem.

Scand. 7, 870 [I953]) separated by paper chromatography, using of water-saturated sec-butanol, containing 3 O / o glacial acetic acid and a trace of KCN as a developer, extracts from lichens and showed, among other things, the VitaminsBl2, Bl2s and the factor C.

In general, paper chromatography is only used for analytical purposes Purposes and is only used occasionally for preparative purposes; in these cases are either thick cardboard boxes or even columns, consisting of one on top of the other Paper discs, used. Both methods can of course only be special Serve scientific purposes and found until now because of their awkwardness or costliness no general entry into the laboratory; of their application in technology there can be no question for the time being.

It is also known to use different mixtures of substances on chromatography columns to separate, which consist of cellulose pulp (cf. H. G. Cassidy, »Adsorption and Chromatography ", New York 195I) or from cellulose powder (J. N. Balston and B. E. Talbot, "Aguideto filter paper and cellulose powder chromatography," London I952; F. Cramer, "Papierchromatographie", 2nd edition, Heidelberg 1953).

In some summarizing works (see F. Cramer, "Paper Chromatography") is of the opinion that, as a rule, the same Rf values on the column can be obtained as in the usual paper chromatogram. Then it would have to be expected that the separation of vitamins of the Bl2 group on columns, consisting of cellulose, n-Butanol and water, is practically impracticable - in analogy to the paper chromatographic Experience. Contrary to this assumption, the most surprising and unexpected Observation made that when using appropriately prepared chromatography columns from cellulose powder or cellulose flakes, n-butanol and water as well as from water-saturated n-Butanol (with the addition of cyanide ions if necessary) is an excellent developer Separation of vitamins of the Bl2 group at very high speed of the zones can be achieved. Apart from the great sharpness of the zones, the new chromatographic method relative R-values R-Wet - shift of the band lowering the liquid column above the column). achieves that in none of the previously known paper chromatographic or chromatographic methods for separating the vitamin B12 components were attainable. The new observation is completely surprising because the Separation of the same mixtures of the vitamin Bl2 group using paper chromatography Method under Using the same developer totally fails (which agrees with the state of the art based on the above literature). The fact of the very different behavior of the same mixtures of vitamin Bl2 factors in partition chromatographic separation on paper and in the cellulose column if the same developer is used, this can be explained by the fact that it is we are dealing here with two different types of separation mechanisms.

I. In the separation by paper chromatography, the solvent moves in paper thanks to the capillary forces of the cellulose fiber. This will presumably the two components of the solvent mixture are not absorbed to the same extent, so that the composition of the developer in the paper is different from that in the stock liquid.

2. In column chromatography, according to the invention, the cellulose mixed with enough n-butanol and water before filling into the column. at no capillary forces come into play during the development of the chromatogram, no segregation of the developer can therefore take place.

It is of course possible that other factors make up the difference are responsible in the effectiveness of these two methods. It appears anyway completely excluded that differences in the cellulose quality are responsible for this are, because column chromatography could be used to purify practically any well and not too degraded type of cellulose - both from cotton and from Wood - the same effects can be achieved.

An attempt was made by varying the working conditions at the Paper chromatography of vitamin B12 factors increase the separating effect in the Assumption that by partially saturating the paper strips with water similar conditions achieved and similar separation effects are created as in column chromatography. As can be seen from Table I, these attempts failed. Even with saturation the paper strips with salts could not use any of the column chromatography according to the invention approximately the same effects can be achieved.

These experiments seem to have sufficiently proven that column chromatography is different in essence from paper chromatography and that under none Test conditions an approximation of the two methods is possible.

The results of these tests are summarized in Table I. As can be seen from this table, by increasing the humidity level the paper strip no effect can be achieved. By saturating the paper strips with phosphate buffer of pH 7.0 a faster migration of the zones could be achieved but they were very indistinct and partly merged into one another. A transfer of this working technique to the cellulose column would be - apart from the inadequate separation - due to the need to use buffering Salts are practically worthless, as the salts are difficult to separate from the vitamin B12 factors are. The separability of the vitamin B12 factors I and II with the help of sec-butanol is relatively good when using untreated paper; the factors III In general, however, and V cannot be separated easily; on with phosphate buffer Factors III and V can be separated from the PH 7.0 impregnated paper, however, the zones are much less sharp than without the use of salts.

Table I Paper Chromatography of a Mixture of Vitamin Bl2 Factors I, II, III and V, obtained from digested sludge.

Whatman I paper. Temperature 18-20 ° C. Run Rf values of the factors Pretreatment of the paper Time developer Characteristics of the zones Hours I paper time 1 i II III + V not pretreated, air dry 48 n-butanol, 0.03 not separated, no zones very saturated with RF value Water + 0.005 01o 'low NaCN (+) immersed in water, then I hour 48 the same. o, o58 0.025; about 3 mm factor I well separated hiked at 200 partially dried, not separates in a 0.66 molar solution of 48 the same. No separation, bands no zones NIl PO4 (pH 4.6) immersed, hiked II mm then dried for 24 hours at 20 ° in a 0.66 molar phosphate 24 like 0.225 0, I58 1 0, I20 0, OI5 diffusely smeared, pH 7.0 buffer immersed, zones one inside the other then running dry for 1 hour at 200 (t) applied as an aqueous solution of PH 7.0 Run Rf values of the factors Pretreatment of the paper Time developer Characteristics of the zones Hrs. I (II 1 III V not pretreated, air dry 24 secondary 0.29 o, 23 0, 1555 sharp zones, however Butanol, gel III and V do not saturates with separated Water +0.005 0Io NaCN (+) smeared into a 0.66 molar solution of 48 similar spots, no zones KH2PO4 (pH 4.6) submerged, hiked about 4.5 cm then dried at 200 for 24 hours in a 0.66 molar phosphate 24 like 0.42 0.34 0.27 o, I2 factor V strongly diffuse buffer from pil 7.0 submerged, smeared, something then I hrs at 200 dried with sharper zones I to III (+) applied as an aqueous solution of pH 7.0 Experiments with cellulose columns using different alcohols or ketones on the one hand and different types of cellulose on the other hand showed that only n-butanol and partly also sec-butanol were used to separate the vitamin Bl2 factors are suitable. Alcohols and ketones that are completely miscible with water - mixed with different amounts of water - result in no or very diffuse zones. Benzyl alcohol, amyl alcohol and methyl ethyl ketone are unsuitable because of their limited water absorption.

From higher alcohols or ketones plus lower alcohols one can Prepare mixtures whose water absorption capacity is that of n-butanol is similar and therefore give about the same separation effects as n-butanol. The usage However, solvent mixtures have no advantages over n-butanol.

Of the types of cellulose tested, both those made of cotton are suitable as well as those made from wood pulp. There are certain differences in effectiveness apparently due to the degree of purity and the different aftertreatment.

The separation effects achieved with the help of sec-butanol in the cellulose column exceeded the corresponding paper chromatographic results. The separation However, the B12 components III and V were still inadequate, as was the case in the following experiment shows: 5 g of cellulose powder were mixed with 50 ccm of cyanide-containing aqueous sec-butanol shaken and left to stand overnight. This mixture was then used in the next Filled into a column in the manner described in more detail below. Served as developer a mixture of 160 cc sec-butanol, 35 cc water and 5 cc of an o, z0 / o aqueous NaCN solution of pA 7.0. The following R values were observed during development: Vitamin B1 factor R value I 0.35 - 0.46 II 0.11-0.18 III 0.04 - 0.07 V 0.03 - 0.05 A comparison of these values with the corresponding values in Table I shows that Using sec-butanol, the column causes a better separation effect than the paper. As a result of the insufficient differences in the R values of the last two However, here the Bl2 factors III and V merge into one another.

In contrast, the partition chromatographic separation of the vitamin B12 factors revealed with the help of cellulose columns using n-butanol-water as a developer excellent results far better than using any other tested alcohols and which therefore present the basis for the method according to the invention, the follow-up d is described in detail.

Developer. 100 cc of n-butanol are mixed with 25 cc of a z. B. o.20 / oigen Sodium cyanide solution of p, 7.0 mixed and then with non-carbonated water up shaken to saturation. The developer is then left standing or by filtration clarified. n-butanol, which contains smaller amounts of water, e.g. B. IoO / o is suitable as a result of the much slower development less than water-saturated n-butanol.

Preparation of the column. There are z. B. 50 g cellulose powder with 500 cc of n-butanol, saturated with non-carbonated water, mixed. This mixture must contain at least as much cyanide ions as the cellulose to adsorb able.

Then, with vigorous shaking or stirring, z. B. 20 ccm of non-carbonated water was added. It is shaken or stirred for so long until the material is completely homogeneous. After standing for several hours, the cellulose pulp is ready to use.

The adsorptive saturation of the cellulose with cyanide ions is next the maintenance of a neutral pH value is necessary for all vitamin Bt2 factors always to be obtained in the same complex form, so that it is defined and reproducible R values can be achieved.

When using cellulose flakes, more water-saturated is produced accordingly n-butanol is used, otherwise the mass will be too thick.

The column to be filled, which is usually closed at the bottom with a tap is partially filled with the developer solution. Then it is in the column the n-butanol cellulose pulp through a funnel while opening the Drain cock introduced. During the settling of the cellulose flakes, the column becomes always rotated around its vertical axis so that the filling takes place evenly. The cellulose pulp is poured in until the settled filling mass is approximately twice the height of the desired column, e.g. B.

14 cm, and the liquid level 2 to 3 cm above the cellulose mass lies. A suitable filter disc is now inserted into the column (without the air bubble get under the disc) and with the help of a suitable tamper the cellulose column is compressed to t / 2 to 2/3 of the original height. As soon the developer has drained so far that the liquid level has almost disappeared the valve is closed and the material to be chromatographed is released in water-saturated n-butanol or applied to a dry material such as z. B. Kieselguhr, filled into the tube through a funnel. If the material was applied as a dry product, it is mixed with a little water-saturated n-butanol moistened and after placing a second piece of filter paper with the help of a The rammer slightly squeezed. After opening the tap are now by adding very small amounts of developer bring the vitamin B12 factors into the actual column flushed, after which development can begin.

Course of the chromatography. It has been proven through numerous attempts especially with pure vitamins of the Bi2 group, determined that the order of Vitamin B12 in development in the cellulose column is the same as factors in the paper chromatogram or on the aluminum oxide column. In chromatography a mixture of factors I and II is preceded by factor I (see Fig. I) a mixture of factors III and V, factor III (see Fig. 2). With numerous Chromatograms of factors I, II, III, and V were determined for each factor characteristic R values observed: vitamin B12 factor R value I 0.40 - 0.59 II 0.21 - 0.29 III 0.08-0.123 V 0.04 - 0.059 The fluctuations in the R values are on due to the different manufacturing methods of the column, as it is not possible is to undertake this process in an absolutely similar manner. While the relative R values are largely independent of the water content of the column, the absolute R values depend strongly depends on the amount of water stirred in. It is therefore advisable to always stir the same amount of water into the cellulose pulp, namely that amount of Water with which the optimal selectivity of the vitamin B12 factors can be achieved. A series of experiments in which 5 g of cellulose powder and 50 ccm of n-butanol are saturated with water, with the addition of various additional amounts of water showed that the optimal amount of water is between 1 and 3 cc. Without Tests carried out with additional water resulted in diffuse zones. If more than 3 ccm of water the cellulose powder forms agglomerations that are difficult to remove, whereby the zones are also blurred. Reduce the absolute R-values with increasing addition of water.

The advantages of the outlined inventive chromatographic The main methods are as follows: I. A simple manufacture of the chromatography column, 2. A short development period. Factors I and II can be used in 5 to 6 hours developed and eluted. The factors III to V take I2 to I8 hours.

3. An unusually high degree of selectivity. With careful implementation and if the contamination with salts or organic foreign matter is not too great In a chromatogram, a practically quantitative separation of the components can be achieved I, II, III and V can be achieved (see Fig. 3) 4. Cheap and easy to regenerate of the developer. After extracting the vitamins of the Bt2 group from the eluate with water the butyl alcohol can be used again after a simple distillation.

5. A low need for developers. To carry out the chromatography extremely small amounts of water-saturated n-butanol are required, which also from Fig. I to 3 can be seen.

The abscissa shows the volume of developer required, the ordinate the amount of vitamin B12 factor eluted; the latter value is for the factors II and III can be calculated using the following formula: Vitamin B12 factor ~ optical Density at 36I m, in gamma / ccm = oho207 The separation of the different vitamin B, 2 types from digested sludge using the adsorption chromatographic method is only available in certain embodiment feasible, namely especially using Aluminum oxide in the presence of cyanide ions in the pH range 6 to 8, especially below Use of mixtures of acetone with water by changing the concentration of water gradually increases in the course of the development of the chromatogram. Despite the usefulness However, this method has the following in comparison with the method according to the invention Disadvantages: I. A relatively laborious manufacture of the adsorption column, 2. A long development period. A chromatogram often takes several days.

3. A relatively low selectivity.

Only factor I can be used in a chromatogram practically be separated quantitatively. Other factors must be rechromatographed.

4. High losses of developer (e.g. acetone) through evaporation. More difficult to regenerate the developer (e.g. dehydration of acetone).

5. The consumption of developer is a multiple of the volume of water-saturated n-butanol in the process according to the invention.

It must be expressly emphasized that in the process described, whose disadvantages compared to the method according to the invention have been discussed, an adsorbent such as aluminum oxide is used, so that it is this is an adsorption chromatographic method in which the vitamins of the B12 group are adsorbed on the column and eluted by the developer. In contrast, the method according to the invention describes a distribution chromatographic method Method in which there is no adsorption of the vitamin B12 factors on the column material takes place, but rather a distribution of vitamins of the Bl2 group between the water retained in the cellulose fiber and the eluent flowing past. The vitamins of the Bl2 group are not adsorbed on cellulose.

Distribution chromatographic methods for the separation of vitamins of the B1 group in chromatography columns have so far only been used sporadically. Thus, E. Lester Smith et al. (Proc.

Biochem. Soc. from May 29, 1948; Spring .. proc. 9, No. I [1950]) den Antiperniziosafactor with the help of silica gel columns, using them as a developer too two-thirds of the water-saturated n-butanol, alone or with the addition of phenol, were used. Even when using n-propanol or

Isopropanol containing 10 to 20% water, satisfactory chromatograms. The same authors also used starch in place of silica gel. They watched on starch columns R values of 0.5 and 1.2 for vitamins B12b and Bl2. K. H. Fantes et al. (Proc. Roy. Soc.

B I36, 592, [I950]) used to develop the silica gel distribution chromatograms n-butanol containing II to I2 O / o water. These authors also described experiments with silica gel columns using mixtures of n-butanol and phenol or Cresol instead of pure n-butanol.

They found that these blends had no appreciable advantages over them have n-butanol, except for a somewhat faster elution. When using mixtures distribution chromatograms were obtained from n-propanol or isopropanol and 10 to 25% water as developer obtained that were very similar to those achievable with n-butanol-water.

Louis Lewis, U. J. et al. (J. Biol. Chem. I94, 539, cm9521) the vitamin Bl2-active material obtained from faeces with the help of silica gel columns using water-saturated butanol as a developer The vitamin Bl2 factors, that were held at the top of the column (that were not developed), the authors removed together with the silica gel and eluted the active material from it with 650% ethanol. J. E. Ford and J. W. G. Porter (Biochem. J. 51, Proc. V [1952]) The vitamins of the Bl2 group obtained from calf faeces were chromatographed on moist Silica gel using water-saturated sec-butanol, containing a trace of cyanide. With the help of this method, they separated the mixture of vitamin B12 factors in two fractions, one of which contained vitamin B12 and factor B, the others the factor A, but not in a completely pure state.

The partition chromatographic experiments with starch columns showed that this material is used to separate mixtures of vitamin B12 factors Digested sludge is not suitable. Several types of starch have been made using different ones Investigated developer, with always very diffuse chromatograms without zone formation were obtained.

Even silica gel columns completely allowed when using with water Miscible alcohols with the usual content of water and cyanide do not separate of mixtures of vitamin B12 factors from digested sludge. Not much better behaved in this case water-saturated sec-butanol. A partial separation succeeded when using water-saturated n-butanol, but only when using very large volumes of developer and development time of several days. It was in an example of a purified concentrate of vitamins obtained from digested sludge the Bl2 group with the help of a silica gel column using water-saturated Chromatographed n-butanol and cyanide as developers, in principle according to the instructions by E. Lester Smith et al. (Biochem. J. 52, 389 [1952]). As can be seen from Fig. 4 is, in this way the vitamins of the Bl2 group could be divided into four fractions (I to 4) be split. In order to more accurately identify these fractions were the eluates are combined in fractions and, after appropriate concentration, with the aid of the process according to the invention is chromatographed on cellulose-n-butanol-water columns. The result is shown in Table 2.

From Table 2 it can be seen that with the help of the silica gel column obtained fractions, with the exception of fraction I, contaminated with the neighboring fractions was. With the help of the method according to the invention, however, the fractions I to 4 can be cleaned with ease.

A repetition of the chromatography also gave completely homogeneous zones.

The new method according to the invention is explained in more detail by the following example: Example A pre-purified mixture of vitamin B12 factors obtained from digested sludge, deposited on diatomaceous earth and dried, is applied to a column which, as described above, consists of cellulose powder, n-butanol and water with the addition of the usual amount of Table 2, partition chromatography of fractions I to 4 (from silica gel chromatography) on a cellulose-n-butanol-water column. Fraction distribution chromatogram in cellulose-n-butanol-water the with the help of the R-value Silica gel column zone color ll R-value t identified factor I strong purple 0.45 1 2 strong red o, 24 II very faint red o, og III 3 very weak J red o, 26 II strong red o, og III 4 strong red '0.08 III strong j violet 0.04 V Sodium cyanide was produced; Column diameter 11.5 mm, height 70 mm.

After development with water-saturated n-butanol containing the usual Amount of sodium cyanide, the individual vitamin Bl2 factors are in practically completely separated form in the eluate. The factors II and III can then without further Purification can be brought to crystallization (see Fig. 3).

Fig. 1 shows the partition chromatography of a mixture of the vitamin B12 factors I and II from digested sludge using a column made of cellulose powder, n-butanol and water with the addition of cyanide. Developer: water-saturated and C N'-containing n-butanol, column diameter 11.5 mm, height 70 mm; Fig. 2 shows the partition chromatography a mixture of vitamin Bl., - factors III and V from digested sludge; Test conditions like Fig. I; Fig. 3 shows the partition chromatography of a mixture of the vitamin B12 factors I, II, III and V from digested sludge; Test conditions as in Fig. I; Fig. 4 shows the Partition chromatography of a purified digested sludge concentrate using a Column made of silica gel, n-butanol and water with the addition of cyanide. Developer: water-saturated and C N'-containing n-butanol.

Column diameter 26 mm, height 200 mm.

Claims (6)

PATENT CLAIMS: I. Process for partition chromatographic Purification and separation of the vitamins of the BI group, characterized in that for Production of the partition chromatographic columns cellulose pulp and as a developer aqueous n-butanol can be used. 2. The method according to claim I, characterized in that as a developer n-Butanol with a water content of at least IO O / o is used. 3. The method according to claims I and 2, characterized in that that the developer is a very small amount, e.g. B. 0, OOI mol / l of cyanide ions. 4. Process according to Claims I to 3, characterized in that that cotton cellulose or wood cellulose is used in the form of powder or flakes will. 5. The method according to claims I to 4, characterized in that that the cellulose material with water-saturated n-butanol and water to a homogeneous The pulp is stirred and then poured into the chromatography column. 6. The method according to claims I to 5, characterized in that that the cellulose pulp used to produce the chromatography column with cyanide ions is adsorptively saturated.